TW202144345A - Kras mutant protein inhibitors - Google Patents
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- TW202144345A TW202144345A TW110109246A TW110109246A TW202144345A TW 202144345 A TW202144345 A TW 202144345A TW 110109246 A TW110109246 A TW 110109246A TW 110109246 A TW110109246 A TW 110109246A TW 202144345 A TW202144345 A TW 202144345A
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- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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Abstract
Description
本發明涉及KRAS突變蛋白抑制劑、含有該抑制劑的組合物及其用途。The present invention relates to KRAS mutein inhibitors, compositions containing the same, and uses thereof.
RAS代表一組189個胺基酸的單體球狀蛋白質(21kDa分子量)，其與質膜相連，並且其結合GDP或GTP任一者。RAS起著分子開關的作用。當RAS含有結合的GDP時，其處於休眠或關閉位置並且是「無活性的」。當細胞暴露於某些促進生長的刺激物時，RAS被誘導將其結合的GDP交換為GTP。在結合GTP的情形下，RAS被「開啟」，並且能夠與其它蛋白質(其「下游靶標」)相互作用並將它們激活。RAS蛋白本身將GTP水解回GDP從而使其自身變成關閉狀態的固有能力非常低。關閉RAS需要被稱作GTP酶激活蛋白(GAP)的外源蛋白，其與RAS相互作用，並大大地加快GTP向GDP的轉化。RAS中影響其與GAP相互作用或將GTP轉化回GDP的能力的任何突變都將導致蛋白質的長時間激活，並因此向細胞發出信號，告訴其繼續生長和***。因為這些訊號導致細胞生長和***，所以過度活躍的RAS訊號傳導可能最終會導致癌症。RAS represents a group of 189 amino acid monomeric globular proteins (21 kDa molecular weight) that are attached to the plasma membrane and that bind either GDP or GTP. RAS acts as a molecular switch. When RAS contains bound GDP, it is in a dormant or closed position and is "inactive". When cells are exposed to certain growth-promoting stimuli, RAS is induced to exchange its bound GDP for GTP. In the context of binding GTP, RAS is "turned on" and is able to interact with other proteins (its "downstream targets") and activate them. The inherent ability of the RAS protein itself to hydrolyze GTP back to GDP thereby turning itself into the off state is very low. Shutting down RAS requires an exogenous protein called GTPase activating protein (GAP), which interacts with RAS and greatly accelerates the conversion of GTP to GDP. Any mutation in RAS that affects its ability to interact with GAP or convert GTP back to GDP will result in prolonged activation of the protein and thus signal the cell to continue growing and dividing. Because these signals cause cells to grow and divide, overactive RAS signaling may eventually lead to cancer.
在結構上，RAS蛋白含有G結構域，所述結構域負責RAS的酶促活性、鳥嘌呤核苷酸結合及水解(GTP酶反應)。其還含有被稱為CAAX盒的C末端延伸區，所述延伸區可以被翻譯後修飾，並且負責將蛋白質靶向細胞膜。G結構域其含有磷酸結合環(P環)。P環代表核苷酸在蛋白質中結合的袋，並且這是帶有保守胺基酸殘基的結構域的剛性部分，所述保守胺基酸殘基是核苷酸結合及水解所必需的(甘氨酸12和賴胺酸16)。G結構域還含有被稱為開關I區(殘基30-40)和開關II(殘基60-76)區，這兩者都是蛋白質的動態部分，因為該動態部分在靜態和加載狀態之間轉化，其常常被表示為「彈簧加載」機制。主要的相互作用是由蘇胺酸-35和甘胺酸-60與GTP的γ-磷酸酯形成的氫鍵，它們分別維持開關I區和開關II區處於其活性構象。在GTP水解和磷酸酯釋放後，這兩者鬆弛成非活性GDP構象。Structurally, RAS proteins contain a G domain, which is responsible for the enzymatic activity of RAS, guanine nucleotide binding and hydrolysis (GTPase reaction). It also contains a C-terminal extension called the CAAX box, which can be post-translationally modified and is responsible for targeting proteins to the cell membrane. G domain It contains a phosphate binding loop (P loop). The P-loop represents the pocket in which nucleotides bind in proteins, and this is the rigid part of the domain with conserved amino acid residues necessary for nucleotide binding and hydrolysis ( Glycine 12 and Lysine 16). The G domain also contains regions known as switch I (residues 30-40) and switch II (residues 60-76), both of which are the dynamic part of the protein because the dynamic part is between the static and loaded states conversion, which is often denoted as a "spring-loaded" mechanism. The main interaction is the hydrogen bond formed by threonine-35 and glycine-60 with the γ-phosphate of GTP, which maintain switch I and switch II regions, respectively, in their active conformations. After GTP hydrolysis and phosphate release, both relax to the inactive GDP conformation.
RAS亞家族中最著名的成員是HRAS、KRAS和NRAS，它們主要與許多類型的癌症有關。RAS基因的三種主要亞型(HRAS、NRAS或KRAS)中的任一者的突變都是人類腫瘤形成中最常見的事件。發現約30％的所有人類腫瘤中的RAS基因攜帶一些突變。值得注意的是，在25-30％的腫瘤中檢測到KRAS突變。相比之下，在NRAS和HRAS家族成員中致癌突變的發生率要低得多(分別為8％和3％)。最常見的KRAS突變見於P環中的殘基G12和G13處以及殘基Q61處。The best-known members of the RAS subfamily are HRAS, KRAS, and NRAS, which are primarily associated with many types of cancer. Mutations in any of the three major isoforms of the RAS gene (HRAS, NRAS or KRAS) are the most common events in human tumorigenesis. The RAS gene was found to carry some mutation in about 30% of all human tumors. Notably, KRAS mutations are detected in 25-30% of tumors. In contrast, the incidence of oncogenic mutations was much lower in NRAS and HRAS family members (8% and 3%, respectively). The most common KRAS mutations are found in the P-loop at residues G12 and G13 and at residue Q61.
G12C是KRAS基因的頻繁突變(甘胺酸-12突變為半胱胺酸)。已經在約13％的癌症發生、約43％的肺癌發生以及在幾乎100％的MYH相關息肉病(家族性結腸癌症候群)中發現了這種突變。然而，用小分子靶向該基因具有挑戰性。G12C is a frequent mutation of the KRAS gene (glycine-12 to cysteine). This mutation has been found in about 13% of cancers, about 43% of lung cancers, and in almost 100% of MYH-associated polyposis (familial colon cancer syndrome). However, targeting this gene with small molecules is challenging.
因此，儘管在此領域中已取得進展，但本領域中仍然需要用於例如通過抑制KRAS、HRAS或NRAS來治療癌症的改進的化合物及方法。本發明滿足此需求並提供其他的相關優勢。Therefore, despite the progress made in this field, there remains a need in the art for improved compounds and methods for treating cancer, eg, by inhibiting KRAS, HRAS or NRAS. The present invention meets this need and provides other related advantages.
一方面，本發明提供式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽：
在一些實施方式中，本發明提供式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽：
在一些實施方式中，R11 、R12 、R13 、R14 或R15 獨立地選自-OH；-F；-Cl；-Br；-NRa Rb ；-C1-3 烷基；-OC1-3 烷基；-C1-3 亞烷基-OH；-C1-3 亞烷基-O-C1-3 烷基；被-F或-Cl取代的-C1-3 烷基；被-F或-Cl取代的-O-C1-3 烷基；-SO2 Ra ；-CN；-C(=O)NRa Rb ；-C(=O)Ra ；-OC(=O)Ra ；-C(=O)ORa ；3元碳環基；4元碳環基；5元碳環基或6元碳環基； Ra 或Rb 獨立地選自氫或-C1-3 烷基。In some embodiments, R 11 , R 12 , R 13 , R 14 , or R 15 are independently selected from -OH; -F; -Cl; -Br; -NR a R b ; -C 1-3 alkyl; -OC 1-3 alkyl; -C 1-3 alkylene-OH; -C 1-3 alkylene-OC 1-3 alkyl; -C 1-3 alkyl substituted by -F or -Cl ; -OC 1-3 alkyl substituted by -F or -Cl; -SO 2 R a ; -CN; -C(=O)NR a R b ;-C(=O)R a ;-OC(= O)R a ; -C(=O)OR a ; 3-membered carbocyclyl; 4-membered carbocyclyl; 5-membered carbocyclyl or 6-membered carbocyclyl; R a or R b are independently selected from hydrogen or - C 1-3 alkyl.
在一些實施方式中，R11
、R12
、R13
、R14
或R15
獨立地選自-OH、-F、-Cl、-NH2
、-NHCH3
、-N(CH3
)2
、-CH3
、-CH2
CH3
、-CH2
CH2
CH3
、-CH(CH3
)2
、-OCH3
、-OCH2
CH3
、-OCH2
CH2
CH3
、-OCH(CH3
)2
、-CH2
OH、-CH2
CH2
OH、-CH2
OCH3
、-CHF2
、-CH2
F、-CF3
、-OCH2
F、-OCHF2
、-OCF3
、-SO2
CH3
、-CN、-C(=O)NH2
、-C(=O)CH3
、-OC(=O)CH3
、-C(=O)OCH3
或3元碳環基。In some embodiments, R 11 , R 12 , R 13 , R 14 , or R 15 are independently selected from -OH, -F, -Cl, -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , - CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 OCH 3, -CHF 2, -CH 2 F, -
在一些實施方式中，R11 、R12 、R13 、R14 或R15 獨立地選自-OH、-F、-Cl、-NH2 、-CH3 或-CF3 。In some embodiments, R 11 , R 12 , R 13 , R 14 or R 15 are independently selected from -OH, -F, -Cl, -NH 2 , -CH 3 or -CF 3 .
在一些實施方式中，R11 選自-OH或-NH2 ；且R12 、R13 、R14 或R15 獨立地選自-F或-Cl。In some embodiments, R 11 is selected from -OH or -NH 2 ; and R 12 , R 13 , R 14 or R 15 are independently selected from -F or -Cl.
在一些實施方式中，R1
選自：
在一些實施方式中，R21 選自氫；-F；-Cl；-Br；-C1-3 烷基；被-F或-Cl的-C1-3 烷基；-C2-3 烯基；或-C3-6 碳環基。In some embodiments, R 21 is selected from hydrogen; -F; -Cl; -Br; -C 1-3 alkyl; a is -F or -Cl -C 1-3 alkyl; -C 2-3 alkenyl or -C 3-6 carbocyclyl.
在一些實施方式中，R21 選自氫；-F；-Cl；甲基；乙基；丙基；異丙基；被-F取代的甲基；被-F取代的乙基；被-F取代的丙基；被-F取代的異丙基；乙烯基；丙烯基；3元碳環基；4元碳環基；5元碳環基；或6元碳環基。In some embodiments, R 21 is selected from hydrogen; -F; -Cl; methyl; ethyl; propyl; isopropyl; methyl substituted with -F; ethyl substituted with -F; substituted propyl; isopropyl substituted with -F; vinyl; propenyl; 3-membered carbocyclyl; 4-membered carbocyclyl; 5-membered carbocyclyl; or 6-membered carbocyclyl.
在一些實施方式中，R21 選自-F或-Cl。In some embodiments, R 21 is selected from -F or -Cl.
在一些實施方式中，R21 選自-F。In some embodiments, R 21 is selected from -F.
在一些實施方式中，R22 選自氫；-F；-Cl；-Br；-C1-3 烷基；被-F或-Cl取代的-C1-3 烷基；-C2-3 烯基；或-C3-6 碳環基。In some embodiments, R 22 is selected from hydrogen; -F; -Cl; -Br; -C 1-3 alkyl; substituted by -F or -Cl is -C 1-3 alkyl; -C 2-3 Alkenyl; or -C 3-6 carbocyclyl.
在一些實施方式中，R22 選自氫；-F；-Cl；甲基；乙基；丙基；異丙基；被-F取代的甲基；被-F取代的乙基；被-F取代的丙基；被-F取代的異丙基；乙烯基；丙烯基；3元碳環基；4元碳環基；5元碳環基；或6元碳環基。In some embodiments, R 22 is selected from hydrogen; -F; -Cl; methyl; ethyl; propyl; isopropyl; methyl substituted with -F; ethyl substituted with -F; substituted propyl; isopropyl substituted with -F; vinyl; propenyl; 3-membered carbocyclyl; 4-membered carbocyclyl; 5-membered carbocyclyl; or 6-membered carbocyclyl.
在一些實施方式中，R22 是氫。In some embodiments, R 22 is hydrogen.
在一些實施方式中，R3 選自苯基、萘基、5元雜芳基、6元雜芳基、7元雜芳基、8元雜芳基、9元雜芳基或10元雜芳基，所述的雜芳基在每次出現時獨立地包含1、2或3個選自N或O的雜原子，所述的苯基、萘基、5元雜芳基、6元雜芳基、7元雜芳基、8元雜芳基、9元雜芳基或10元雜芳基獨立地任選地被1個R31 、2個R31 、3個R31 、4個R31 或5個R31 取代或不取代。In some embodiments, R 3 is selected from phenyl, naphthyl, 5-membered heteroaryl group, a 6-membered heteroaryl group, 7-membered heteroaryl group, 8-membered heteroaryl, 9- or 10-membered heteroaryl aryl, heteroaryl base, said heteroaryl group independently at each occurrence contains 1, 2 or 3 heteroatoms selected from N or O, said phenyl, naphthyl, 5-membered heteroaryl, 6-membered heteroaryl radical, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl, or 10-membered heteroaryl independently optionally replaced by 1 R 31 , 2 R 31 , 3 R 31 , 4 R 31 or 5 R 31 substituted or unsubstituted.
在一些實施方式中，R3 選自苯基或6元雜芳基，所述的雜芳基包含1個2選自N的雜原子，所述的苯基或所述的6元雜芳基在每次出現時獨立地任選地被1個R31 、2個R31 、3個R31 或4個R31 取代或不取代。In some embodiments, R 3 is selected from phenyl or 6-membered heteroaryl, the heteroaryl contains 1 heteroatom selected from N, the phenyl or the 6-membered heteroaryl Each occurrence is independently optionally substituted or unsubstituted with 1 R 31 , 2 R 31 , 3 R 31 , or 4 R 31 .
在一些實施方式中，R3
選自
在一些實施方式中，每個R31 在每次出現時獨立地選自-F、-Cl、-Br、-C1-3 烷基、-CN、-OH、-O-C1-3 烷基、-NH2 、-NH(C1-3 烷基)、-N(C1-3 烷基)2 或-C3-6 碳環基。In some embodiments, each R 31 at each occurrence is independently selected from -F, -Cl, -Br, -C 1-3 alkyl, -CN, -OH, -OC 1-3 alkyl, -NH 2 , -NH(C 1-3 alkyl), -N(C 1-3 alkyl) 2 or -C 3-6 carbocyclyl.
在一些實施方式中，每個R31 在每次出現時獨立地選自-F、-Cl、甲基、乙基、丙基、異丙基、-CN、-OH、甲氧基、乙氧基、丙氧基、異丙氧基、-NH2 、-NHCH3 、-NHCH2 CH3 、-NH(CH2 CH2 CH3 )、-NH(CH(CH3 )2 )、-N(CH3 )2 、-N(CH2 CH3 )2 、-N(CH3 )(CH2 CH3 )、3元碳環基、4元碳環基、5元碳環基或6元碳環基。In some embodiments, each R 31 at each occurrence is independently selected from -F, -Cl, methyl, ethyl, propyl, isopropyl, -CN, -OH, methoxy, ethoxy group, propoxy, isopropoxy, -NH 2 , -NHCH 3 , -NHCH 2 CH 3 , -NH(CH 2 CH 2 CH 3 ), -NH(CH(CH 3 ) 2 ), -N( CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), 3-membered carbocyclyl, 4-membered carbocyclyl, 5-membered carbocyclyl, or 6-membered carbocyclyl base.
在一些實施方式中，每個R31 在每次出現時獨立地選自甲基或異丙基。In some embodiments, each R 31 at each occurrence is independently selected from methyl or isopropyl.
在一些實施方式中，R3
選自
在一些實施方式中，R3
是
在一些實施方式中，R4
選自
在一些實施方式中，R4
選自
在一些實施方式中，R4
選自
在一些實施方式中，R41
選自
在一些實施方式中，R4a 、R4b 或R4c 獨立地選自氫、-F、-Cl、甲基、乙基、丙基、異丙基、-CH2 -N(CH3 )2 、-CH2 -N(CH2 CH3 )2 或-CH2 -N(CH3 )(CH2 CH3 )。In some embodiments, R 4a , R 4b or R 4c is independently selected from hydrogen, -F, -Cl, methyl, ethyl, propyl, isopropyl, -CH 2 -N(CH 3 ) 2 , -CH 2 -N (CH 2 CH 3 ) 2 or -CH 2 -N (CH 3) ( CH 2 CH 3).
在一些實施方式中，R4a 、R4b 或R4c 獨立地選自氫、-F、甲基或-CH2 -N(CH3 )2 。In some embodiments, R 4a , R 4b or R 4c is independently selected from hydrogen, -F, methyl, or -CH 2 -N(CH 3 ) 2 .
在一些實施方式中，R4a選自氫或-F； R4b 是氫； R4c 選自氫或-CH2 -N(CH3 )2 。In some embodiments, R4a is selected from hydrogen or -F; R 4b is hydrogen; R 4c is selected from hydrogen or -CH 2 -N (CH 3) 2 .
在一些實施方式中，R41
獨立地選自：
在一些實施方式中，R4
選自
在一些實施方式中，每個R42 在每次出現時獨立地選自-C1-3 烷基；-C1-3 亞烷基-CN；或被-F或-Cl取代的-C1-3 烷基。In some embodiments, each R 42 is independently selected at each occurrence from -C 1-3 alkyl; -C 1-3 alkylene-CN; or -C 1 substituted with -F or -Cl -3 alkyl.
在一些實施方式中，每個R42 在每次出現時獨立地選自甲基；乙基；丙基；異丙基；-亞甲基-CN；-亞乙基-CN；-亞丙基-CN；被-F取代的甲基；被-F取代的乙基；被-F取代的丙基；或被-F取代的異丙基。In some embodiments, each R42 is independently selected at each occurrence from methyl; ethyl; propyl; isopropyl; -methylene-CN; -ethylene-CN; -propylene -CN; methyl substituted with -F; ethyl substituted with -F; propyl substituted with -F; or isopropyl substituted with -F.
在一些實施方式中，每個R42 在每次出現時獨立地選自甲基；乙基；-亞甲基-CN或被-F取代的甲基。In some embodiments, each R 42 is independently selected at each occurrence from methyl; ethyl; -methylene-CN or methyl substituted with -F.
在一些實施方式中，每個R42 在每次出現時獨立地選自-CH3 、-CH2 CH3 、-CH2 CN、-CHF2 或-CF3 。In some embodiments, each R 42 is independently selected at each occurrence from -CH 3 , -CH 2 CH 3 , -CH 2 CN, -CHF 2 or -CF 3 .
在一些實施方式中，R4
選自：
在一些實施方式中，R4
選自：
在一些實施方式中，R4
選自：
在一些實施方式中，所述的化合物選自：
另一方面，本發明提供製備式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽，所述方法包括過渡金屬鈀或鎳試劑催化的根據如下反應方案1的式（II）的化合物與式（III）的化合物之間的偶聯反應或根據如下反應方案2的式（II’）的化合物與式（III’）的化合物之間的偶聯反應：
方案1：
另一方面，本發明提供式（IV）的中間體、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽：
在一些實施方式中，所述的式(IV)的中間體為式(IV’)：
在一些實施方式中，所述的式(IV)的中間體選自：
另一方面，本發明提供製備本發明的式（I’）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽的方法，所述的方法包括在鹼性條件下的根據如下反應方案3的式（IV’）的中間體與式（V’）的化合物之間的反應：
方案3：
另一方面，本發明提供藥物組合物，其包括本發明所述的式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽，及至少一種藥學上可接受的賦形劑。在一些實施方式中，所述的化合物與所述的賦形劑的重量比的範圍為約0.0001至約10。在一些實施方式中，所述的化合物與所述的賦形劑的重量比的範圍為約0.01至約0.8。在一些實施方式中，所述的化合物與所述的賦形劑的重量比的範圍為約0.02至約0.2。在一些實施方式中，所述的化合物與所述的賦形劑的重量比的範圍為約0.05至0.15。In another aspect, the present invention provides a pharmaceutical composition comprising the compound of formula (I) described in the present invention, its stereoisomers, its atropisomers, its pharmaceutically acceptable salts, its stereoisomers A pharmaceutically acceptable salt of the compound or a pharmaceutically acceptable salt of an atropisomer thereof, and at least one pharmaceutically acceptable excipient. In some embodiments, the weight ratio of the compound to the excipient ranges from about 0.0001 to about 10. In some embodiments, the weight ratio of the compound to the excipient ranges from about 0.01 to about 0.8. In some embodiments, the weight ratio of the compound to the excipient ranges from about 0.02 to about 0.2. In some embodiments, the weight ratio of the compound to the excipient ranges from about 0.05 to 0.15.
另一方面，本發明提供本發明所述的式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽或本發明所述的藥物組合物在製備治療與KRAS G12C突變蛋白相關的癌症的藥物中的應用。在一些實施方式中，所述的癌症選自血液癌、胰腺癌、結腸癌、直腸癌、結直腸癌或肺癌。在一些實施方式中，所述的血液癌選自急性髓性白血病或急性淋巴性白血病；所述的肺癌選自非小細胞肺癌或小細胞肺癌。In another aspect, the present invention provides the compound of formula (I), its stereoisomer, its atropisomer, its pharmaceutically acceptable salt, and its stereoisomer according to the present invention, which are pharmacologically acceptable. Use of the accepted salt or the pharmaceutically acceptable salt of its atropisomer or the pharmaceutical composition of the present invention in the manufacture of a medicament for treating cancer associated with KRAS G12C mutein. In some embodiments, the cancer is selected from blood cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, or lung cancer. In some embodiments, the blood cancer is selected from acute myeloid leukemia or acute lymphoblastic leukemia; the lung cancer is selected from non-small cell lung cancer or small cell lung cancer.
另一方面，提供一種治療患有與KRAS G12C突變蛋白相關的癌症受試者的方法，所述方法包括向所述受試者給予治療有效劑量的本發明所述的式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽或本發明所述的藥物組合物。在一些實施方式中，所述的癌症選自血液癌、胰腺癌、結腸癌、直腸癌、結直腸癌或肺癌。在一些實施方式中，所述的血液癌選自急性髓性白血病或急性淋巴性白血病；所述的肺癌選自非小細胞肺癌或小細胞肺癌。In another aspect, there is provided a method of treating a subject suffering from a cancer associated with a KRAS G12C mutein, the method comprising administering to the subject a therapeutically effective dose of a compound of formula (I) of the present invention, Its stereoisomer, its atropisomer, its pharmaceutically acceptable salt, its stereoisomer's pharmaceutically acceptable salt or its atropisomer's pharmaceutically acceptable salt or the present The pharmaceutical composition of the invention. In some embodiments, the cancer is selected from blood cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, or lung cancer. In some embodiments, the blood cancer is selected from acute myeloid leukemia or acute lymphoblastic leukemia; the lung cancer is selected from non-small cell lung cancer or small cell lung cancer.
另一方面，本發明提供用於治療與KRAS G12C突變蛋白相關的癌症的本發明的所述的式（I）的化合物、其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥效上可接受的鹽或其阻轉異構體的藥學上可接受的鹽或本發明所述的藥物組合物。在一些實施方式中，所述的癌症選自血液癌、胰腺癌、結腸癌、直腸癌、結直腸癌或肺癌。在一些實施方式中，所述的血液癌選自急性髓性白血病或急性淋巴性白血病；所述的肺癌選自非小細胞肺癌或小細胞肺癌。In another aspect, the present invention provides the compound of formula (I) of the present invention, its stereoisomer, its atropisomer, its pharmaceutically acceptable for use in the treatment of cancer associated with KRAS G12C muteins , the pharmaceutically acceptable salt of its stereoisomer or the pharmaceutically acceptable salt of its atropisomer or the pharmaceutical composition of the present invention. In some embodiments, the cancer is selected from blood cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, or lung cancer. In some embodiments, the blood cancer is selected from acute myeloid leukemia or acute lymphoblastic leukemia; the lung cancer is selected from non-small cell lung cancer or small cell lung cancer.
定義definition
除非另有說明，本文所用的術語「鹵素」（“halogen” 或 “halo”）是指氟、氯、溴或碘。優選的鹵素基團包括-F、-Cl和-Br。更優選地的鹵素為-F或-Cl。The term "halogen" ("halogen" or "halo") as used herein refers to fluorine, chlorine, bromine or iodine, unless otherwise specified. Preferred halogen groups include -F, -Cl and -Br. More preferred halogens are -F or -Cl.
除非另有說明，本文所用的術語「烷基」包括具有直鏈或支鏈的飽和一價烴基。例如，烷基包括甲基、乙基、丙基、異丙基、正丁基、異丁基、第二丁基、第三丁基、正戊基、3-(2-甲基)丁基、2-戊基、2-甲基丁基、新戊基、正己基、2-己基和2-甲基戊基。類似地，C1-6 烷基中的C1-6 被定義為直鏈或支鏈排列中該基團具有1、2、3、4、5或6個碳原子。Unless otherwise specified, the term "alkyl" as used herein includes saturated monovalent hydrocarbon radicals having straight or branched chains. For example, alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-(2-methyl)butyl , 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl and 2-methylpentyl. Similarly, C 1-6 alkyl C 1-6 is defined as having 1,2,3,4,5 or 6 carbon atoms, a straight-chain or branched-chain arrangement of the group.
術語「亞烷基」是指通過從如上定義的烷基除去氫原子而獲得的雙官能團。例如，亞甲基（即-CH2 -）、亞乙基（即-CH2 -CH2 -或-CH(CH3 )-）和亞丙基（即-CH2 -CH2 -CH2 -、-CH(-CH2 -CH3 ))-或-CH2 -CH(CH3 )-）。The term "alkylene" refers to a bifunctional group obtained by removing a hydrogen atom from an alkyl group as defined above. For example, methylene (ie -CH 2 -), ethylene (ie -CH 2 -CH 2 - or -CH(CH 3 )-) and propylene (ie -CH 2 -CH 2 -CH 2 - , -CH(-CH 2 -CH 3 ))- or -CH 2 -CH(CH 3 )-).
術語「烯基」是指含有一個或多個雙鍵的直鏈或支鏈烴基，通常長度為2至20個碳原子。例如，「C2-6 鏈烯基」含有2至6個碳原子。例如，烯基包括但不限於例如乙烯基、丙烯基、丁烯基、2-甲基-2-丁烯-1-基、庚烯基、辛烯基等。The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing one or more double bonds, usually 2 to 20 carbon atoms in length. For example, "C 2-6 alkenyl" contains 2 to 6 carbon atoms. For example, alkenyl groups include, but are not limited to, eg, vinyl, propenyl, butenyl, 2-methyl-2-buten-1-yl, heptenyl, octenyl, and the like.
術語「炔基」含有一個或多個三鍵的直鏈或支鏈烴基，通常長度為2至20個碳原子。例如，「C2-6 炔基」含有2至6個碳原子。代表性的炔基包括但不限於，例如，乙炔基、1-丙炔基、1-丁炔基、庚炔基、辛炔基等。The term "alkynyl" contains a straight or branched chain hydrocarbon group containing one or more triple bonds, usually 2 to 20 carbon atoms in length. For example, "C 2-6 alkynyl" contains 2 to 6 carbon atoms. Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl, and the like.
術語「烷氧基」基團是由前述烷基形成的氧醚。The term "alkoxy" group is an oxygen ether formed from the aforementioned alkyl groups.
除非另有說明，本文所用的術語「芳基」或「芳環」是指僅含有環碳原子的未取代或取代的單環或多環芳環系統。優選的芳基是單環或雙環6-10元芳環系統。苯基和萘基是優選的芳基。最優選的芳基是苯基。As used herein, unless otherwise indicated, the term "aryl" or "aromatic ring" refers to an unsubstituted or substituted monocyclic or polycyclic aromatic ring system containing only ring carbon atoms. Preferred aryl groups are monocyclic or bicyclic 6-10 membered aromatic ring systems. Phenyl and naphthyl are preferred aryl groups. The most preferred aryl group is phenyl.
除非另有說明，本文所用的術語「雜環基」或「雜環」是指及含有一個或多個（如2、3、4、5或6個）環雜原子的未取代和取代的單環或多環非芳族環系，其包括單環的雜環（基）、雙環的雜環（基）、橋環的雜環（基）、稠環的雜環（基）及螺環的雜環（基）。優選的雜原子包括N、O和S，包括N-氧化物、硫氧化物和二氧化物。優選地，該雜環（基）是完全飽和的或具有一個或多個不飽和度的三至十元環。雜環基或雜環的定義包括多個取代度（優選一個、兩個或三個取代度）。這種雜環基的實例包括但不限於氮雜環丁烷基、吡咯烷基、哌啶基、哌嗪基、氧代哌嗪基、氧代哌啶基、氧代氮雜環庚基、氮雜環庚基、四氫呋喃基、二氧戊環基、四氫咪唑基、四氫噻唑基、四氫噁唑基、四氫吡喃基、嗎福林基、硫代嗎福林基、硫代嗎福林基亞碸、硫代嗎福林基碸或噁二唑基。Unless otherwise specified, the terms "heterocyclyl" or "heterocycle" as used herein refer to unsubstituted and substituted monocycles containing one or more (eg, 2, 3, 4, 5 or 6) ring heteroatoms Cyclic or polycyclic non-aromatic ring systems, including monocyclic heterocycle (radical), bicyclic heterocycle (radical), bridged ring heterocycle (radical), fused ring heterocycle (radical) and spirocyclic heterocycle (radical) Heterocycle (base). Preferred heteroatoms include N, O and S, including N-oxides, sulfur oxides and dioxides. Preferably, the heterocycle (radical) is a fully saturated or three to ten membered ring with one or more degrees of unsaturation. The definition of heterocyclyl or heterocycle includes multiple degrees of substitution (preferably one, two or three). Examples of such heterocyclyl groups include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxoazepanyl, Azacycloheptyl, tetrahydrofuranyl, dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, sulfur On behalf of the moflyn base, thiomorphine base or oxadiazolyl.
除非另有說明，本文所用的術語「雜芳基」或「雜芳環」表示含有碳和至少一個雜原子的芳環系統。雜芳基或雜芳環可以是單環的或多環的，取代的或未取代的。單環雜芳基在環中可具有1至4個雜原子，而多環雜芳基可包含1至10個雜原子。多環雜芳基環可含有稠合、螺環或橋環結合，例如，雙環雜芳基是多環雜芳基。雙環雜芳基環可含有8至12個成員原子。單環雜芳基環可含有5至8個成員原子（碳原子和雜原子）。雜芳基的實例包括，但不限於噻吩基、呋喃基、咪唑基、異噁唑基、噁唑基、吡唑基、吡咯基、噻唑基、噻二唑基、***基、吡啶基、嗒嗪基、吲哚基、氮雜吲哚基、吲唑基、苯并咪唑基、苯并呋喃基、苯并噻吩基、苯并異噁唑基、苯并噁唑基、苯并吡唑基、苯并噻唑基、苯并噻二唑基、苯并***基、腺嘌呤基、喹啉基或異喹啉基。Unless otherwise indicated, the terms "heteroaryl" or "heteroaromatic ring" as used herein refer to an aromatic ring system containing carbon and at least one heteroatom. Heteroaryl groups or heteroaromatic rings can be monocyclic or polycyclic, substituted or unsubstituted. Monocyclic heteroaryl groups can have 1 to 4 heteroatoms in the ring, while polycyclic heteroaryl groups can contain 1 to 10 heteroatoms. Polycyclic heteroaryl rings may contain fused, spiro or bridged ring linkages, eg, a bicyclic heteroaryl is a polycyclic heteroaryl. Bicyclic heteroaryl rings may contain from 8 to 12 member atoms. Monocyclic heteroaryl rings may contain from 5 to 8 member atoms (carbon and heteroatoms). Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, pyridyl, Pyridazinyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzopyrazole group, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, adeninyl, quinolinyl or isoquinolinyl.
術語「碳環基」或「碳環」是指取代或未取代的單環、雙環、橋環、稠環或螺環的僅包含碳原子的非芳族飽和環，「碳環」或「碳環基」包括不帶取代度的「環烷基」及帶有一個或多個取代度的碳環基或碳環。示例性的「碳環基」包括但不限於環丙基、環丁基、環戊基、環己基等。The term "carbocyclyl" or "carbocycle" refers to a substituted or unsubstituted monocyclic, bicyclic, bridged, fused or spirocyclic non-aromatic saturated ring containing only carbon atoms, "carbocycle" or "carbon" "Cyclic" includes "cycloalkyl" with no degree of substitution and carbocyclyl or carbocycle with one or more degrees of substitution. Exemplary "carbocyclyl groups" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
術語「氧代」是指氧與所連接的碳原子一起形成
術語「-C1-6 亞烷基-N(C1-6 烷基)2 」是指被-N(C1-6 烷基)2 取代的如上定義的-C1-6 烷基。The term "-C 1-6 alkylene-N(C 1-6 alkyl) 2 " refers to -C 1-6 alkyl as defined above substituted with -N(C 1-6 alkyl) 2 .
術語「-C1-6 亞烷基-CN」是指被-CN取代的如上定義的-C1-6 烷基。The term "-C 1-6 alkylene-CN" refers to a -C 1-6 alkyl group as defined above substituted with -CN.
術語「雜烷基」是指如上定義的烷基中的任意兩個碳原子之間存在雜原子，例如N原子或O原子，例如「雜C2-6 烷基」表示在C2-6 烷基任意兩個碳原子之間存在N原子或O原子，包括但不限於-CH2 OCH3 、-CH2 CH2 OCH3 、-CH2 NHCH3 或-CH2 N(CH3 )2 等。The term "heteroalkyl" refers to the presence of a heteroatom, such as a N atom or an O atom, between any two carbon atoms in an alkyl group as defined above, for example " heteroC2-6alkyl " means a C2-6alkane There is an N atom or an O atom between any two carbon atoms of the radical, including but not limited to -CH 2 OCH 3 , -CH 2 CH 2 OCH 3 , -CH 2 NHCH 3 or -CH 2 N(CH 3 ) 2 and the like.
如本文所用，術語「組合物」旨在涵蓋包含特定量的特定成分的產品，以及直接或間接由特定量的特定成分的組合產生的任何產品。因此，含有本發明化合物作為活性成分的藥物組合物以及製備本發明化合物的方法也是本發明的一部分。As used herein, the term "composition" is intended to encompass products comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Accordingly, pharmaceutical compositions containing the compounds of the present invention as active ingredients and methods of preparing the compounds of the present invention are also part of the present invention.
本發明所述的化合物可以藥學上可接受的鹽的形式存在。為了藥學用途，本發明所述化合物的鹽指非毒的「藥學上可接受的鹽」。藥學上可接受的鹽的形式包括藥學上可接受的酸/陰離子或堿/陽離子鹽。藥學上可接受的酸/陰離子鹽通常採用一種形式，該形式中鹼性氮原子被無機酸或有機酸質子化。The compounds of the present invention may exist in the form of pharmaceutically acceptable salts. For pharmaceutical use, salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". Pharmaceutically acceptable salt forms include pharmaceutically acceptable acid/anion or phosphonium/cation salts. Pharmaceutically acceptable acid/anion salts generally take a form in which the basic nitrogen atom is protonated with an inorganic or organic acid.
本發明在其範圍內包括本發明化合物的前藥。通常，這種前藥是化合物的功能性衍生物，其易於在體內轉化為所需化合物。因此，在本發明的治療方法中，術語「給藥」應包括用特定公開的化合物，或用可能未具體公開的化合物，但給予受試者後在體內轉化為特定化合物的化合物治療描述的各種病症。用於選擇和製備合適的前藥衍生物的常規方法描述於例如「前藥設計」（“Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.）。The present invention includes within its scope prodrugs of the compounds of the present invention. Typically, such prodrugs are functional derivatives of compounds that are readily converted to the desired compound in vivo. Thus, in the methods of treatment of the present invention, the term "administering" shall include treatment of the various described compounds with a specifically disclosed compound, or with a compound that may not be specifically disclosed, but which upon administration to a subject converts to the specified compound in vivo disease. Conventional methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrugs" ("Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.).
分子中特定位置的任何取代基或變量的定義旨在獨立于該分子中其他位置的取代基或變量的定義。應當理解，本發明所屬技術領域具通常知識者可以選擇本發明化合物上的取代基和取代模式，以提供化學穩定的化合物，並且可以通過本領域已知的技術以及本文闡明的方法容易地合成。The definition of any substituent or variable at a particular position in a molecule is intended to be independent of the definition of substituents or variables at other positions in that molecule. It will be appreciated that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to provide chemically stable compounds that can be readily synthesized by techniques known in the art and by the methods set forth herein.
本發明包括所述化合物可含有一個或多個不對稱中心，因此可產生非對映異構體和光學異構體。本發明包括所有這些可能的非對映異構體及其外消旋混合物、它們基本上純的拆分的對映異構體、所有可能的幾何異構體、及其藥學上可接受的鹽。The present invention contemplates that the compounds may contain one or more asymmetric centers and thus may give rise to diastereomers and optical isomers. The present invention includes all such possible diastereomers and racemic mixtures thereof, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof .
本發明包括化合物的所有立體異構體及其藥學上可接受的鹽。此外，還包括立體異構體的混合物以及分離的特定立體異構體。在用於製備這些化合物的合成方法的過程中，或在使用本領域技術人員已知的外消旋化或差向異構化方法的過程中，這些方法的產物可以是立體異構體的混合物。The present invention includes all stereoisomers of the compounds and their pharmaceutically acceptable salts. In addition, mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of the synthetic methods used to prepare these compounds, or during the use of racemization or epimerization methods known to those skilled in the art, the products of these methods may be mixtures of stereoisomers .
本發明所述「立體異構體」是指分子中原子或原子團互相連接次序相同，但空間排列不同而引起的異構體，其包括構型異構體和構象異構體，其中的構型異構體又包括幾何異構體和旋光異構體，旋光異構體主要包括對映異構體和非對映異構體。The term "stereoisomers" in the present invention refers to isomers caused by atoms or atomic groups in the molecule in the same order of interconnection, but with different spatial arrangements, including configurational isomers and conformational isomers. Isomers include geometric isomers and optical isomers, and optical isomers mainly include enantiomers and diastereomers.
本文提供的某些化合物可以作為阻轉異構體存在，其為構象立體異構體，當由於與分子的其他部分的空間相互作用而阻止或大大減緩圍繞分子中單鍵的旋轉時出現。本文提供的化合物包括所有的阻轉異構體，包括純的單獨的阻轉異構體，各自的富集的阻轉異構體或各自的非特異性混合物。如果圍繞單鍵的旋轉勢壘足夠高，並且構象之間的相互轉化足夠慢，則可以允許阻轉異構體的分離。Certain compounds provided herein can exist as atropisomers, which are conformational stereoisomers that occur when rotation about a single bond in a molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule. The compounds provided herein include all atropisomers, including pure individual atropisomers, respective enriched atropisomers or respective nonspecific mixtures. If the rotational barrier around the single bond is high enough and the interconversion between conformations is slow enough, separation of atropisomers can be allowed.
本發明旨在包括本發明化合物中存在的所有原子同位素。同位素是具有相同原子序數但質量數不同的原子。作為一般實例而非限制，氫的同位素包括氘和氚。氫的同位素可表示為1 H（氫），2 H（氘）和3 H（氚）。它們通常也表示為D（氘）和T（氚）。在本申請中，CD3 表示甲基，其中所有氫原子都是氘。碳的同位素包括13 C和14 C。使用適當的同位素標記的試劑代替非標記試劑，本發明的同位素標記的化合物通常可以通過本領域技術人員已知的常規技術或通過與本文所述類似的方法製備。The present invention is intended to include all atomic isotopes present in the compounds of the present invention. Isotopes are atoms with the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of hydrogen can be represented as 1 H (hydrogen), 2 H (deuterium) and 3 H (tritium). They are also commonly denoted D (deuterium) and T (tritium). In this application, CD 3 represents a methyl group in which all hydrogen atoms are deuterium. Isotopes of carbon include 13 C and 14 C. Using an appropriate isotopically-labeled reagent in place of a non-labeled reagent, the isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described herein.
除非另有說明，當式（I）化合物的互變異構體存在時，本發明包括任何可能的互變異構體及其藥學上可接受的鹽，以及它們的混合物。Unless otherwise indicated, when tautomers of compounds of formula (I) exist, the present invention includes any possible tautomers and pharmaceutically acceptable salts thereof, as well as mixtures thereof.
本發明的藥物組合物包含本發明所述的化合物（其立體異構體、其阻轉異構體、其藥學上可接受的鹽、其立體異構體的藥學上可接受的鹽或其阻轉異構體的藥效上可接受的鹽）作為活性成分，以及藥學上可接受的載體和任選的其它的佐劑。儘管在任何給定情況下最合適的途徑將取決於特定的宿主，以及為病症（為治療該病症而正在施用該活性成分）的性質和嚴重程度，但該組合物包括適於口服、直腸、局部和腸胃外（包括皮下、肌肉內和靜脈內）給藥的組合物。藥物組合物可以方便地以單位劑型存在，並通過藥學領域熟知的任何方法製備。The pharmaceutical composition of the present invention comprises the compound of the present invention (its stereoisomers, its atropisomers, its pharmaceutically acceptable salts, its pharmaceutically acceptable salts of stereoisomers or its hindered isomers). a pharmaceutically acceptable salt of the isomer) as the active ingredient, together with a pharmaceutically acceptable carrier and optionally other adjuvants. Although the most appropriate route in any given situation will depend on the particular host, as well as the nature and severity of the condition for which the active ingredient is being administered, the compositions include oral, rectal, Compositions for topical and parenteral (including subcutaneous, intramuscular and intravenous) administration. The pharmaceutical compositions may conveniently be presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
實踐中，根據常規藥物配製技術，本發明的式（I）代表的化合物或其前藥或代謝物或其藥學上可接受的鹽可以作為活性成分與藥物載體組合成緊密混合物（intimate admixture）。載體可以採取多種形式，這取決於給藥途徑所需的製劑形式，例如口服或腸胃外（包括靜脈內）給藥途徑。因此，本發明的藥物組合物可以作為適於口服給藥的離散單位存在，例如每個含有預定量的活性成分的膠囊、扁囊劑（cachets）或片劑。此外，組合物可以粉末形式、顆粒形式、溶液形式、水性液體中的懸浮液、非水液體、水包油乳液或油包水乳液形式存在。除了上述常見劑型外，式（I）代表的化合物或其藥學上可接受的鹽還可以通過控釋裝置和/或遞送裝置給藥。該組合物可以通過任何藥學方法製備。通常，這些方法包括使活性成分與構成一種或多種必需成分的載體結合的步驟。通常，通過將活性成分與液體載體或細碎的固體載體或兩者均勻且緊密地混合來製備組合物。然後可以方便地將產品成形為所需的樣式。In practice, the compound represented by formula (I) of the present invention, or a prodrug or metabolite thereof, or a pharmaceutically acceptable salt thereof, can be combined as an active ingredient in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a variety of forms, depending on the desired form of formulation for the route of administration, eg, oral or parenteral (including intravenous) routes of administration. Accordingly, the pharmaceutical compositions of the present invention may be presented as discrete units suitable for oral administration, such as capsules, cachets or tablets each containing a predetermined quantity of the active ingredient. In addition, the compositions may exist in powder form, granular form, solution form, suspension in aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil emulsion. In addition to the above-mentioned common dosage forms, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can also be administered by a controlled release device and/or a delivery device. The composition can be prepared by any method of pharmacy. Generally, these methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. Generally, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers, or both. The product can then be conveniently shaped into the desired style.
所用的藥物載體/賦形劑可以例如是固體、液體或氣體。固體載體的實例包括乳糖、石膏粉、蔗糖、滑石粉、明膠、瓊脂、果膠、***膠、硬脂酸鎂和硬脂酸。液體載體的實例是糖漿、花生油、橄欖油和水。氣態載體的實例包括二氧化碳和氮氣。在製備用於口服劑型的組合物中，可以使用任何方便的藥物介質。例如，水、乙二醇、油、醇、調味劑、防腐劑、著色劑等可被用於形成例如懸浮液、酏劑和溶液的口服液體製劑；而澱粉、糖、微晶纖維素、稀釋劑、造粒劑、潤滑劑、粘合劑、崩解劑等載體可被用於形成如粉末、膠囊和片劑的口服固體製劑。由於易於給藥，片劑和膠囊是優選的口服劑量單位，其中使用固體藥物載體。任選地，片劑可以通過標準水性或非水性技術包衣。The pharmaceutical carrier/excipient used can be, for example, solid, liquid or gaseous. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen. In preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like can be used to form oral liquid preparations such as suspensions, elixirs, and solutions; while starch, sugar, microcrystalline cellulose, dilute Carriers such as agents, granulating agents, lubricants, binders, disintegrating agents, etc., can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units in which solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or non-aqueous techniques.
含有本發明組合物的片劑可以通過壓製或模塑製備，任選地含有一種或多種輔助成分或佐劑。壓縮片劑可以通過在合適的機器中壓製如粉末或顆粒自由流動形式的活性成分，任選地與粘合劑、潤滑劑、惰性稀釋劑、表面活性劑或分散劑混合來製備。模製片劑可以通過在合適的機器中模製用惰性液體稀釋劑潤濕的粉末化合物的混合物來製備。每片優選含有約0.05mg至約5g活性成分，每個扁囊劑或膠囊優選含有約0.05mg至約5g活性成分。例如，用於人口服給藥的製劑可含有與適當和方便量的載體物質混合的約0.5mg至約5g活性劑，載體物質可占總組合物的約0.05％至約95％。單位劑型通常含有約0.01mg至約2g活性成分，通常0.01mg、0.02mg、1mg、2mg、3mg、4mg、5mg、6mg、7mg、8mg、9mg、10mg、25mg、50mg、 100mg、200mg、300mg、400mg、500mg、600mg、800mg、1000mg、1500mg或2000mg。Tablets containing the compositions of the present invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient, and each cachet or capsule preferably contains from about 0.05 mg to about 5 g of the active ingredient. For example, formulations for oral administration to humans may contain from about 0.5 mg to about 5 g of active agent in admixture with a suitable and convenient amount of carrier material, which may comprise from about 0.05% to about 95% of the total composition. A unit dosage form typically contains from about 0.01 mg to about 2 g of active ingredient, usually 0.01 mg, 0.02 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400mg, 500mg, 600mg, 800mg, 1000mg, 1500mg or 2000mg.
適合腸胃外給藥的本發明藥物組合物可以製備成活性化合物在水中的溶液或懸浮液。可以包括合適的表面活性劑，例如羥丙基纖維素。分散體也可以在甘油、液體聚乙二醇及其在油中的混合物中製備。此外，可以包含防腐劑以防止微生物的有害生長。Pharmaceutical compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of the active compounds in water. Suitable surfactants such as hydroxypropyl cellulose may be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. In addition, preservatives may be included to prevent harmful growth of microorganisms.
適用於注射用途的本發明藥物組合物包括無菌水溶液或分散液。此外，組合物可以是用於臨時製備這種無菌可注射溶液或分散體的無菌粉末的形式。在所有情況下，最終的可注射形式必須是無菌的並且必須是有效流動的以便於注射。藥物組合物在製造和儲存條件下必須是穩定的；因此，優選應該防止如細菌和真菌的微生物的污染下保藏。載體可以是例如含有水、乙醇、多元醇（例如甘油、丙二醇和液體聚乙二醇）、植物油及其合適的混合物的溶劑或分散介質。Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Additionally, the compositions may be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be fluid for easy injection. Pharmaceutical compositions must be stable under the conditions of manufacture and storage; therefore, they should preferably be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
本發明的藥物組合物可以是適於局部使用的形式，例如氣溶膠、乳膏、軟膏、洗劑、撒粉等。此外，組合物可以是適用於透皮裝置的形式。利用本發明的化合物或其藥學上可接受的鹽，這些製劑可以通過常規加工方法來製備。例如，通過將親水性材料和水與約0.05wt％至約10wt％的化合物混合以產生具有所需稠度的乳膏或軟膏來製備乳膏或軟膏。The pharmaceutical compositions of the present invention may be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, dusting powder, and the like. Furthermore, the compositions may be in a form suitable for use in transdermal devices. These formulations can be prepared by conventional processing methods utilizing a compound of the present invention or a pharmaceutically acceptable salt thereof. For example, a cream or ointment is prepared by mixing a hydrophilic material and water with about 0.05% to about 10% by weight of the compound to produce a cream or ointment of the desired consistency.
本發明的藥物組合物可以是適於直腸給藥的形式，其中載體是固體。優選混合物形成單位劑量栓劑。合適的載體包括可可脂和本領域常用的其他材料。栓劑可以通過首先將組合物與軟化或熔化的載體混合，然後在模具中冷卻和成型來方便地形成。The pharmaceutical compositions of the present invention may be in a form suitable for rectal administration wherein the carrier is a solid. Preferably the mixture is formed into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently formed by first mixing the composition with a softened or molten carrier, then cooling and shaping in a mold.
除了上述載體成分之外，上述藥物製劑可以適當地包括一種或多種另外的載體成分，例如稀釋劑、緩衝劑、調味劑、粘合劑、表面活性劑、增稠劑、潤滑劑、防腐劑（包括抗氧化劑）等等。此外，可以包括其他佐劑以使製劑與預期接受者的血液等滲。含有式I所示化合物或其藥學上可接受的鹽的組合物也可以製備成粉末或液體濃縮物形式。In addition to the aforementioned carrier ingredients, the aforementioned pharmaceutical formulations may suitably include one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surfactants, thickening agents, lubricants, preservatives ( including antioxidants), etc. In addition, other adjuvants may be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of Formula I, or a pharmaceutically acceptable salt thereof, can also be prepared in the form of powders or liquid concentrates.
通常，每天約0.001mg/kg至約150mg/kg體重的劑量水平可用於治療上述病症，或者每位患者每天約0.05mg至約7g。Typically, dosage levels of about 0.001 mg/kg to about 150 mg/kg of body weight per day can be used to treat the above-mentioned conditions, or about 0.05 mg to about 7 g per patient per day.
然而，應理解，任何特定患者的具體劑量水平將取決於包括年齡、體重、一般健康狀況、性別、飲食、給藥時間、給藥途徑、***率、藥物組合以及接受治療的特定疾病的嚴重程度的多種因素。It is to be understood, however, that the specific dosage level for any particular patient will depend on factors including age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease being treated. of multiple factors.
雖然本發明已以實施例揭露如上，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不脫離本發明之精神和範圍內，當可作些許之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。Although the present invention has been disclosed as above with examples, it is not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the appended patent application.
可以使用本文所述的合成方法和反應方案，由市售試劑合成本發明的化合物。概述特定合成路線的示例旨在為本領域的合成化學家提供指導，他們將很容易理解溶劑、濃度、試劑、保護基團、合成步驟的順序、時間、溫度等可以根據需要在本領域技術人員的技術和判斷範圍內進行修改。Compounds of the present invention can be synthesized from commercially available reagents using the synthetic methods and reaction schemes described herein. The examples outlining specific synthetic routes are intended to provide guidance to synthetic chemists in the art, who will readily understand solvents, concentrations, reagents, protecting groups, sequence of synthetic steps, times, temperatures, etc., which can be readily available to those skilled in the art as needed. Modifications are made within the scope of technology and judgment.
提供以下實施例以更好地說明本發明。除非另有明確說明，否則所有份數和百分比均以重量計，所有溫度均為攝氏度。示例中使用了表1中的縮寫：
表1
實施例1
4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物1)；(M)
-4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮；和(P)
-4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 2,6-二氯-5-氟-N-((2-異丙基-4-甲基吡啶-3-基)氨基甲醯)煙醯胺Step 1. 2,6-Dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide
用氮氣置換500-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入2,6-二氯-5-氟煙酸(25.218 g, 120.09 mmol)及SOCl2 (100 mL)。混合物80o C攪拌2 h。將該反應冷卻至室溫並真空濃縮。混合物用CH3 CN稀釋(20 mL)，隨後在0 °C滴加NH3 .H2 O (100 mL)。將混合物在室溫下攪拌1 h。過濾收集固體得25.101 g (粗品)的2,6-二氯-5-氟煙醯胺，其直接用於下一步反應。It was replaced with nitrogen 500-mL round bottom flask and maintain an inert atmosphere of nitrogen, to which was added 2,6-dichloro-5-fluoronicotinic acid (25.218 g, 120.09 mmol) and SOCl 2 (100 mL). 80 o C the mixture was stirred for 2 h. The reaction was cooled to room temperature and concentrated in vacuo. The mixture was diluted with CH 3 CN (20 mL), followed by dropwise addition of 0 ° C and NH 3 .H 2 O (100 mL ). The mixture was stirred at room temperature for 1 h. The solid was collected by filtration to give 25.101 g (crude) of 2,6-dichloro-5-fluoronicotinamide, which was used directly in the next reaction.
用氮氣置換500-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入2,6-二氯-5-氟煙醯胺 (25.101 g, 粗品)及THF (200 mL)。隨後滴加草醯氯 (21.884 g, 172.42 mmol)。將該混合物在80o C攪拌2 h。所得溶液真空濃縮。隨後在0 °C加入2-異丙基-4-甲基吡啶-3-胺(19.830 g, 132.11 mmol)。混合物室溫攪拌1 h。然後加水(200 mL)淬滅該反應。所得溶液用二氯甲烷(3 x 200 mL)萃取，合併有機相並用飽和食鹽水(200 mL)洗滌，無水Na2 SO4 乾燥，殘餘物真空濃縮並加至矽膠柱上，用EA/正己烷(v/v=1:2)洗脫，得到42.01 g (91%)的2,6-二氯-5-氟-N-((2-異丙基-4-甲基吡啶-3-基)氨基甲醯)煙醯胺，其為紅色油。LCMS:m/z = 385 [M+1]+ 。A 500-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which 2,6-dichloro-5-fluoronicotinamide (25.101 g, crude) and THF (200 mL) were added. Oxalyl chloride (21.884 g, 172.42 mmol) was then added dropwise. The mixture was stirred for 2 h at 80 o C. The resulting solution was concentrated in vacuo. 2-Isopropyl-4-methylpyridin-3-amine (19.830 g, 132.11 mmol) was then added at 0 °C. The mixture was stirred at room temperature for 1 h. Water (200 mL) was then added to quench the reaction. The resulting solution was extracted with dichloromethane (3 x 200 mL), the organic phases were combined and washed with saturated brine (200 mL), dried over anhydrous Na 2 SO 4 , the residue was concentrated in vacuo and applied to a silica gel column, washed with EA/n-hexane (v/v=1:2) eluted to give 42.01 g (91%) of 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl ) carbamate) nicotinamide, which is a red oil. LCMS: m/z = 385 [M+1] + .
步驟 2. 7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2,4(1H,3H)-二酮Step 2. 7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidine-2,4(1H,3H)- diketone
用氮氣置換500-mL圓底燒瓶並維持其氮氣氛圍，向其中加入2,6-二氯-5-氟-N-((2-異丙基-4-甲基吡啶-3-基)氨基甲醯)煙醯胺 (42.01 g, 109.83 mmol)及THF (200 mL)並在室溫下攪拌。-10o C加入NaH (9.493 g, 237.33 mmol)。混合物室溫下攪拌2 h。將該反應液傾入水(200 mL)中。所得溶液用二氯甲烷(3 x 100 mL)萃取，合併有機相並用飽和食鹽水(100 mL)洗滌，無水Na2 SO4 乾燥，所得殘餘物真空濃縮並加至矽膠柱上，用EA/正己烷(v/v=61/4)洗脫，得25.11 g (65%)的7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2,4(1H,3H)-二酮，其為紅色油。LCMS:m/z = 349 [M+1]+ 。A 500-mL round-bottom flask was replaced with nitrogen and maintained under nitrogen, to which was added 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl)amino formyl)nicotinamide (42.01 g, 109.83 mmol) and THF (200 mL) and stirred at room temperature. -10 o C was added NaH (9.493 g, 237.33 mmol) . The mixture was stirred at room temperature for 2 h. The reaction solution was poured into water (200 mL). The resulting solution was extracted with dichloromethane (3 x 100 mL), the organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous Na 2 SO 4 , the resulting residue was concentrated in vacuo and applied to a silica gel column, washed with EA/n-hexane Alkane (v/v=61/4) to give 25.11 g (65%) of 7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine [2,3-d]pyrimidine-2,4(1H,3H)-dione as a red oil. LCMS: m/z = 349 [M+1] + .
步驟 3. (S)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯
用氮氣置換500-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2,4(1H,3H)-二酮 (25.074g, 74.96 mmol)、乙腈(200 mL)、三氯氧磷(45.97 g, 299.81 mmol)及DIEA (58.12 g, 449.70 mmol)。混合物80o C攪拌2 h。反應降至室溫並真空濃縮。得4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3- 基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (30 g, 粗品)，其直接用於下一步反應。A 500-mL round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridin[2 ,3-d]pyrimidine-2,4(1H,3H)-dione (25.074 g, 74.96 mmol), acetonitrile (200 mL), phosphorus oxychloride (45.97 g, 299.81 mmol) and DIEA (58.12 g, 449.70 mmol). 80 o C the mixture was stirred for 2 h. The reaction was cooled to room temperature and concentrated in vacuo. to obtain 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (30 g, crude), which was used directly in the next reaction.
用氮氣置換500-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (30 g, 粗品)、乙腈(200 mL)、DIEA (58.12 g, 449.70 mmol)及(S)-3-甲基哌嗪-1-甲酸第三丁酯 (15.801 g, 78.90 mmol)。反應混合物室溫攪拌2 h。然後加水(500 mL)淬滅該反應。所得溶液用乙酸乙酯萃取(3 x 500 mL)，合併有機相並用飽和食鹽水(500 mL)洗滌，無水Na2 SO4 乾燥，過濾並真空濃縮。殘餘物矽膠柱純化，用EA/正己烷(v/v=4/1)洗脫，得15.18 g (38.14% 兩步)的(S)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯，其為黃色固體。LCMS:m/z = 531 [M+1]+ 。A 500-mL round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine Para[2,3-d]pyrimidin-2(1H)-one (30 g, crude), acetonitrile (200 mL), DIEA (58.12 g, 449.70 mmol) and (S)-3-methylpiperazine-1 - tert-butyl formate (15.801 g, 78.90 mmol). The reaction mixture was stirred at room temperature for 2 h. Water (500 mL) was then added to quench the reaction. The resulting solution was extracted with ethyl acetate (3 x 500 mL), the organic phases were combined and washed with saturated brine (500 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column eluting with EA/n-hexane (v/v=4/1) to give 15.18 g (38.14% for two steps) of (S)-4-(7-chloro-6-fluoro-1- (2-Isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperidine tert-butyl oxazine-1-carboxylate as a yellow solid. LCMS: m/z = 531 [M+1] + .
步驟 4. (S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 4. (S)-4-(4-Propenyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridine -3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
用氮氣置換250-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入(S)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯 (14.708 g, 27.698 mmol)、二氯甲烷 (20 mL)及TFA (4 ml)。反應混合物室溫攪拌2 h。將該反應混合物真空濃縮。殘餘物用DCM (20 mL)溶於250-mL圓底燒瓶中，隨後加入DIEA (4 mL)。將反應混合物降至0o C並加入丙烯醯氯(6.756 g, 74.65 mmol)。混合物室溫攪拌1 h，然後加水(100 mL)淬滅該反應。所得溶液用乙酸乙酯萃取(3 x 100 mL)，合併有機相並用飽和食鹽水(100 mL)洗滌，無水Na2 SO4 乾燥，過濾並真空濃縮。殘餘物矽膠柱純化，用EA/hexane(v/v=4/1)洗脫，得16.459 g的(S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為紅色油。LCMS:m/z = 485 [M+1]+ 。A 250-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridine-3) -yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester (14.708 g, 27.698 mmol ), dichloromethane (20 mL) and TFA (4 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo. The residue was dissolved in a 250-mL round bottom flask with DCM (20 mL), followed by the addition of DIEA (4 mL). The reaction mixture was cooled to 0 o C and adding Bing Xixi chloride (6.756 g, 74.65 mmol). The mixture was stirred at room temperature for 1 h, then water (100 mL) was added to quench the reaction. The resulting solution was extracted with ethyl acetate (3 x 100 mL), the organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column eluting with EA/hexane (v/v=4/1) to give 16.459 g of (S)-4-(4-propenyl-2-methylpiperazin-1-yl) -7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as a red oil . LCMS: m/z = 485 [M+1] + .
步驟 5. (S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 5. (S)-4-(4-Propenyl-2-methylpiperazin-1-yl)-7-(5-amino-2,3,4-trifluorophenyl)-6- Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
用氮氣置換20-mL封管並維持其氮氣惰性氛圍，向其中加入(S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (1.485 g, 3.06 mmol)、(5-胺基-2,3,4-三氟苯基)硼酸 (0.773 g, 4.09 mmol)、Pd(PPh3 )4 (0.356 g, 0.31 mmol)、碳酸鈉 (0.372 g, 3.51 mmol)及二氧六環(10 mL)和水(2 mL)。反應混合物80o C攪拌1 h。將該反應混合物過濾並真空濃縮。殘餘物矽膠柱純化，用EA/正己烷(2/1)洗脫，得1.386 g (76%)的(S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-(5-胺基-2,3,4- 三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色固體。LCMS:m/z = 596 [M+1]+ 。A 20-mL tube was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (S)-4-(4-propenyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (1.485 g, 3.06 mmol), (5-amino -2,3,4-Trifluorophenyl)boronic acid (0.773 g, 4.09 mmol), Pd(PPh 3 ) 4 (0.356 g, 0.31 mmol), sodium carbonate (0.372 g, 3.51 mmol) and dioxane ( 10 mL) and water (2 mL). 80 o C the reaction mixture was stirred for 1 h. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by silica gel column eluting with EA/n-hexane (2/1) to give 1.386 g (76%) of (S)-4-(4-propenyl-2-methylpiperazin-1-yl) )-7-(5-amino-2,3,4-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2, 3-d]pyrimidin-2(lH)-one as a yellow solid. LCMS: m/z = 596 [M+1] + .
步驟 6. 4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6- 氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(化合物1)Step 6. 4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl )-6-Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 1)
用氮氣置換100-mL圓底燒瓶並維持其氮氣氛圍，向其中加入(S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(1.444 g, 2.42 mmol)、NCS (1.268 g, 9.50 mmol)和AcOH(20 mL)。混合物室溫攪拌1 d，然後加水(10 mL)淬滅該反應。所得溶液用乙酸乙酯 (3 x 10 mL)萃取，合併有機相並用飽和食鹽水(1 x 50 mL)洗滌，無水Na2 SO4 乾燥，過濾並真空濃縮。殘餘物用Prep-HPLC純化，用CH3 CN/H2 O(v/v=6/4)洗脫，得69 mg (5%)的4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物 1)，其為黃色固體。LCMS:m/z = 630 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.38 – 8.222 (m, 2H), 7.16 (t,J = 4.1 Hz, 1H), 6.87 – 6.60 (m, 1H), 6.23 (d,J = 16.0 Hz, 1H), 5.75 (dd,J = 10.6, 1.8 Hz, 1H), 5.13 – 4.89 (m, 1H),4.59 – 4.26 (m, 2H), 4.22 – 3.94 (m, 1H), 3.92 – 3.44 (m, 3H), 2.75 (s, 1H), 1.93 (d,J = 12.4 Hz, 3H), 1.50 – 1.32 (m, 3H), 1.10 (d,J = 6.4 Hz, 3H), 1.01 – 0.82 (m, 3H)。A 100-mL round bottom flask was replaced with nitrogen and maintained under nitrogen, to which was added (S)-4-(4-propenyl-2-methylpiperazin-1-yl)-7-(5-amino) -2,3,4-Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine-2(1H )-one (1.444 g, 2.42 mmol), NCS (1.268 g, 9.50 mmol) and AcOH (20 mL). The mixture was stirred at room temperature for 1 d, then water (10 mL) was added to quench the reaction. The resulting solution was extracted with ethyl acetate (3 x 10 mL), the organic phases were combined and washed with saturated brine (1 x 50 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by Prep-HPLC, (v / v = 6/4) eluting with CH 3 CN / H 2 O, to give 69 mg (5%) of 4 - ((S) -4- Bingxi Xi-2 -Methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4- Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 1) as a yellow solid. LCMS: m/z = 630 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.38 – 8.222 (m, 2H), 7.16 (t, J = 4.1 Hz, 1H), 6.87 – 6.60 (m, 1H), 6.23 (d, J = 16.0 Hz , 1H), 5.75 (dd, J = 10.6, 1.8 Hz, 1H), 5.13 – 4.89 (m, 1H), 4.59 – 4.26 (m, 2H), 4.22 – 3.94 (m, 1H), 3.92 – 3.44 (m , 3H), 2.75 (s, 1H), 1.93 (d, J = 12.4 Hz, 3H), 1.50 – 1.32 (m, 3H), 1.10 (d, J = 6.4 Hz, 3H), 1.01 – 0.82 (m, 3H).
步驟 7. 4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物1-1) & 4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6- 三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物1-2)Step 7. 4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl )-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (first eluting isomer , compound 1-1) & 4-((S)-4-propenyl-2-methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6- Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second wash Deisomerization, compound 1-2)
採用如下條件，通過Chiral-Prep-HPLC分離純化4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(140 mg)的阻轉異構體混合物：柱子：CHIRALPAK IF, 2cm × 25cm，5um；流動相：((Hex:DCM(v/v=3:1)): IPA(v/v=4:1)； 檢測波長：UV 220nm，得29 mg (20.7%)的4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物1-1)，其為黃色固體。LCMS:m/z = 630 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.48 (t,J = 8.6 Hz, 1H), 8.37 (t,J = 10.0 Hz, 1H), 7.44 (d,J = 5.2 Hz, 1H), 6.84 (td,J = 16.5, 10.9 Hz, 1H), 6.30 (t,J = 15.7 Hz, 1H), 5.88 – 5.77 (m, 1H), 5.06 (s, 1H), 4.49 (dd,J = 38.5, 12.4 Hz, 2H), 4.15 (dd,J = 49.6, 13.5 Hz, 1H), 3.95 – 3.57 (m, 2H), 3.51 – 3.32 (m, 1H), 2.96 (s, 1H), 2.11 (t,J = 14.0 Hz, 3H), 1.51 (s, 3H), 1.24 (d,J = 6.8 Hz, 3H), 1.06 (dd,J = 11.4, 6.9 Hz, 3H)。 和28 mg (20.0%)的4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6- 三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物1-2)，其為黃色固體。LCMS:m/z = 630 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.48 (t,J = 8.6 Hz, 1H), 8.37 (t,J = 10.0 Hz, 1H), 7.44 (d,J = 5.2 Hz, 1H), 6.84 (td,J = 16.5, 10.9 Hz, 1H), 6.30 (t,J = 15.7 Hz, 1H), 5.88 – 5.77 (m, 1H), 5.06 (s, 1H), 4.49 (dd,J = 38.5, 12.4 Hz, 2H), 4.15 (dd,J = 49.6, 13.5 Hz, 1H), 3.95 – 3.57 (m, 2H), 3.51 – 3.32 (m, 1H), 2.96 (s, 1H), 2.11 (t,J = 14.0 Hz, 3H), 1.51 (s, 3H), 1.24 (d,J = 6.8 Hz, 3H), 1.06 (dd,J = 11.4, 6.9 Hz, 3H)。4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(3-amino-2-chloro-4 was isolated and purified by Chiral-Prep-HPLC using the following conditions ,5,6-Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine-2(1H)- Atropisomeric mixture of ketone (140 mg): Column: CHIRALPAK IF, 2cm × 25cm, 5um; Mobile phase: ((Hex:DCM(v/v=3:1)):IPA(v/v=4 : 1); Detection wavelength: UV 220nm, obtained 29 mg (20.7%) of 4-((S)-4-propenyl-2-methylpiperazin-1-yl)-7-(3-amino) -2-Chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine -2(1H)-one (first eluting isomer, compound 1-1) as a yellow solid. LCMS: m/z = 630 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.48 (t, J = 8.6 Hz, 1H), 8.37 (t, J = 10.0 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 6.84 (td, J = 16.5, 10.9 Hz, 1H), 6.30 (t, J = 15.7 Hz, 1H), 5.88 – 5.77 (m, 1H), 5.06 (s, 1H), 4.49 (dd, J = 38.5, 12.4 Hz, 2H), 4.15 (dd, J = 49.6, 13.5 Hz, 1H), 3.95 – 3.57 (m, 2H), 3.51 – 3.32 (m, 1H), 2.96 (s, 1H), 2.11 (t, J = 14.0 Hz, 3H), 1.51 (s, 3H), 1.24 (d, J = 6.8 Hz, 3H), 1.06 (dd, J = 11.4, 6.9 Hz, 3H). and 28 mg (20.0%) of 4-((S)-4-propenyl- 2-Methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4 -Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second eluting isomer, compound 1-2) as a yellow solid. LCMS: m/ z = 630 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.48 (t, J = 8.6 Hz, 1H), 8.37 (t, J = 10.0 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 6.84 (td, J = 16.5, 10.9 Hz, 1H) , 6.30 (t, J = 15.7 Hz, 1H), 5.88 – 5.77 (m, 1H), 5.06 (s, 1H), 4.49 (dd, J = 38.5, 12.4 Hz, 2H), 4.15 (dd, J = 49.6 , 13.5 Hz, 1H), 3.95 – 3.57 (m, 2H), 3.51 – 3.32 (m, 1H), 2.96 (s, 1H), 2.11 (t, J = 14.0 Hz, 3H), 1.51 (s, 3H) , 1.24 (d, J = 6.8 Hz, 3H), 1.06 (dd, J = 11.4, 6.9 Hz, 3H).
實施例 2
4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物 2);(M)
-4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮; 和(P)
-4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物2)Step 1. 4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)- 6-Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidin-2(1H)-one (Compound 2)
用氮氣置換20-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入(S)-4-(4-丙烯醯基-2-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (0.71 g, 1.46 mmol)、(2-胺基-3,4,5,6-四氟苯基)硼酸 (1.512 g, 7.24 mmol)、Pd(PPh3 )4 (0.183 g, 0.16 mmol)、Na2 CO3 (0.491 g, 4.63 mmol)、二氧六環 (8 mL)及水 (2 mL)。反應混合物80o C攪拌1 h。將反應混合物過濾並真空濃縮。殘餘物Prep-HPLC純化，用CH3 CN/H2 O(0.05%NH4 HCO3 )(v:v=2/1)洗脫，得0.181 g (30%)的4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物2)，其為黃色固體。LCMS: m/z =614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.37 (d, J = 4.4 Hz, 1H), 8.28 (s, 1H), 7.21 (s, 1H), 6.76 (d, J = 12.2 Hz, 1H), 6.23 (d, J = 15.6 Hz, 1H), 5.75 (d, J = 10.4 Hz, 1H), 5.16 – 4.83 (m, 2H), 4.64 – 4.20 (m, 2H), 4.20 – 3.89 (m, 1H), 3.89 – 3.40 (m, 2H), 2.77 (s, 1H), 1.95 (s, 3H), 1.57 – 1.28 (m, 3H), 1.28 – 1.02 (m, 3H), 0.93 (d, J = 5.5 Hz, 3H)。A 20-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (S)-4-(4-propenyl-2-methylpiperazin-1-yl)-7-chloro-6- Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (0.71 g, 1.46 mmol), (2-amine yl-3,4,5,6-tetrafluorophenyl)boronic acid (1.512 g, 7.24 mmol), Pd(PPh 3 ) 4 (0.183 g, 0.16 mmol), Na 2 CO 3 (0.491 g, 4.63 mmol), Dioxane (8 mL) and water (2 mL). 80 o C the reaction mixture was stirred for 1 h. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by Prep-HPLC using CH 3 CN / H 2 O ( 0.05% NH 4 HCO 3): eluting with (v v = 2/1) , to give 0.181 g (30%) of 4 - ((S) - 4-Propenyl-2-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2-iso Propyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 2) as a yellow solid. LCMS: m/z =614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.37 (d, J = 4.4 Hz, 1H), 8.28 (s, 1H), 7.21 (s, 1H), 6.76 (d, J = 12.2 Hz, 1H), 6.23 (d, J = 15.6 Hz, 1H), 5.75 (d, J = 10.4 Hz, 1H), 5.16 – 4.83 (m, 2H), 4.64 – 4.20 (m, 2H), 4.20 – 3.89 (m, 1H) , 3.89 – 3.40 (m, 2H), 2.77 (s, 1H), 1.95 (s, 3H), 1.57 – 1.28 (m, 3H), 1.28 – 1.02 (m, 3H), 0.93 (d, J = 5.5 Hz , 3H).
步驟2.(M) -(3S)-4-(7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯Step 2. (M) -(3S)-4-(7-(2-Amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4- Methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester
用氮氣置換反應器並維持其氮氣惰性氛圍，向其中加入(3S )-4-{7-氯-6-氟-(1M )-1-[4-甲基-2-(丙-2-基)吡啶-3-基]-2-氧代-1,2-二氫吡啶駢[2,3-d ]嘧啶-4-基}-3-甲基哌嗪-1-甲酸第三丁酯(1.0 eq) (其在WO2020102730A1的第64頁並被稱為 “PIPAZOLINE”)、Pd(dppf)Cl2 .CH2 Cl2 (0.02 eq)、NaOAc (4.0 eq)和二氧六環(4.0V)。隨後在65-85o C加入H2 O（3.0 V）。然後加入在二氧六環（2.70 V）中的(2-氨基-3,4,5,6-四氟苯基)硼酸（1.15 eq）和H2 O（0.30 V）。將該反應混合物在75±5o C攪拌1h。採用巰基矽膠脫除鈀並用EA、MTBE和庚烷結晶得到(M )-(3S)-4-(7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯，其為黃色固體。LCMS: m/z =660 [M+1]+ 。The reactor was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added ( 3S )-4-{7-chloro-6-fluoro-( 1M )-1-[4-methyl-2-(propane-2 -yl)pyridin-3-yl]-2-oxo-1,2-dihydropyridinepara[2,3- d ]pyrimidin-4-yl}-3-methylpiperazine-1-carboxylic acid tert-butyl ester (1.0 eq) (which is referred to and "PIPAZOLINE" at page 64 of WO2020102730A1), Pd (dppf) Cl 2 .CH 2 Cl 2 (0.02 eq), NaOAc (4.0 eq) and dioxane (4.0 V). H 2 O (3.0 V) was then added at 65-85 o C. Was then added in dioxane (2.70 V) of (2-amino-3,4,5,6-tetrafluorophenyl) borate (1.15 eq) and H 2 O (0.30 V). The reaction mixture was stirred at 75±5 o C for 1 h. Removal of palladium using mercaptosilica and crystallization from EA, MTBE and heptane gave ( M )-(3S)-4-(7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-4-yl)-3- 3-butyl methylpiperazine-1-carboxylate as a yellow solid. LCMS: m/z =660 [M+1] + .
步驟3.(M)
-4-((S)-4-丙烯醯-2-甲基哌嗪-1-基)-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物2-1)
用氮氣置換反應器並維持其氮氣惰性氛圍，向其中加入(M )-(3S)-4-(7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-3-甲基哌嗪-1-甲酸第三丁酯(1.0 eq)和DCM (10 V)。在5±5o C下加入三氟甲磺酸三甲基矽酯(3.5 eq)。將該反應在20±5o C反應1h。用K2 CO3 水溶液和食鹽水洗滌該反應混合物，得到(M )-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-4-((S)-2-甲基哌嗪-1-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮的溶液。將K3 PO4 溶液(5 V, 1.15 eq)加入到該(M )-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-4-((S)-2-甲基哌嗪-1-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮的溶液中。隨後加入在0-10o C加入丙烯醯氯 (1.15 eq)。將反應分離、用5%食鹽水洗滌並在真空下濃縮。粗產品用乙醇重結晶得到(M)-4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡咯駢[2,3-d]嘧啶-2(1H)-酮(化合物2-1) LCMS: m/z =614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.37 (d,J = 4.8 Hz, 1H), 8.39 – 8.25 (m, 1H), 7.20 (d,J = 4.6 Hz, 1H), 6.93 – 6.72 (m, 1H), 6.22 (d,J = 15.9 Hz, 1H), 5.74 (d,J = 10.5 Hz, 1H), 4.92 (s, 1H), 4.57 – 4.27 (m, 2H), 4.26 – 4.02 (m, 1H), 3.79 – 3.49 (m, 2H), 3.35 – 3.23 (m, 1H), 2.85 – 2.70 (m, 1H), 1.94 (s, 3H), 1.55 – 1.34 (m, 3H), 1.10 (d,J = 6.6 Hz, 3H), 0.93 (d,J = 6.5 Hz, 3H)。The reactor was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added ( M )-(3S)-4-(7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-4-yl)-3- 3-butyl methylpiperazine-1-carboxylate (1.0 eq) and DCM (10 V). Trimethylsilyl triflate (3.5 eq) was added at 5±5 oC. The reaction was carried out at 20±5 o C for 1 h. The reaction mixture was washed with K 2 CO 3 solution and brine, to give (M) -7- (2- amino-3,4,5,6-tetrafluoro-phenyl) -6-fluoro-1- (2-isopropyl solution of yl-4-methylpyridin-3-yl)-4-((S)-2-methylpiperazin-1-yl)pyridino[2,3-d]pyrimidin-2(1H)-one . The K 3 PO 4 solution (5 V, 1.15 eq) was added to the (M) -7- (2- amino-3,4,5,6-tetrafluoro-phenyl) -6-fluoro-1- (2- Isopropyl-4-methylpyridin-3-yl)-4-((S)-2-methylpiperazin-1-yl)pyridino[2,3-d]pyrimidin-2(1H)-one in the solution. Followed by addition at 0-10 o C was added Bing Xixi chloride (1.15 eq). The reaction was isolated, washed with 5% brine and concentrated in vacuo. The crude product was recrystallized from ethanol to give (M)-4-((S)-4-propenyl-2-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6 -Tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrrole[2,3-d]pyrimidin-2(1H)-one (Compound 2 -1) LCMS: m/z =614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.37 (d, J = 4.8 Hz, 1H), 8.39 – 8.25 (m, 1H), 7.20 (d, J = 4.6 Hz, 1H), 6.93 – 6.72 (m , 1H), 6.22 (d, J = 15.9 Hz, 1H), 5.74 (d, J = 10.5 Hz, 1H), 4.92 (s, 1H), 4.57 – 4.27 (m, 2H), 4.26 – 4.02 (m, 1H), 3.79 – 3.49 (m, 2H), 3.35 – 3.23 (m, 1H), 2.85 – 2.70 (m, 1H), 1.94 (s, 3H), 1.55 – 1.34 (m, 3H), 1.10 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.5 Hz, 3H).
步驟4.(P) -4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物2-2)Step 4. (P) -4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorobenzene yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 2-2)
除了將步驟2中的(3S )-4-{7-氯-6-氟-(1M )-1-[4-甲基- 2-(丙基-2-基)吡啶-3-基]-2-氧代-1,2-二氫吡啶駢[2,3-d ]嘧啶-4-基}-3-甲基哌嗪-1-甲酸第三丁酯替換為(3S )-4-{7-氯-6-氟-(1P )-1-[4-甲基-2-(丙基-2-基)吡啶-3-基]-2-氧代-1,2-二氫吡啶駢[2,3-d ]嘧啶-4-基}-3-甲基哌嗪-1-甲酸第三丁酯以外，採用如化合物2-1的製備基本相同的步驟得到(P) -4-((S)-4-丙烯醯基-2-甲基哌嗪-1-基)-7-(2-氨基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物2-2)。LCMS:m/z =614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.46 (d,J = 5.0 Hz, 1H), 8.42 – 8.33 (m, 1H), 7.33 – 7.25 (m, 1H), 6.91 – 6.76 (m, 1H), 6.32 (dd,J = 16.7,4.7 Hz, 1H), 5.84 (dd,J = 10.6,1.9 Hz, 1H), 5.28 – 5.08 (m, 1H), 4.63 – 4.36 (m, 2H), 4.24 – 4.03 (m, 1H), 3.96 – 3.79 (m, 1H), 3.79 – 3.54 (m, 1H), 3.45 – 3.35 (m, 0.5H), 3.26 – 3.15 (m, 0.5H),2.89 – 2.76 (m, 1H), 2.12 – 1.99 (m, 3H), 1.46 (d,J = 6.6 Hz, 3H), 1.19 (d,J = 6.8 Hz, 3H), 1.02 (d,J = 6.7 Hz, 3H)。Except that in Step 2 (3 S) -4- {7- chloro-6 - (1 M) -1- [4- methyl --2- (propyl-2-yl) pyridin-3-yl ] -2-oxo-1,2-dihydropyridine-parallel [2,3- d] pyrimidin-4-yl} -3-methyl-piperazine-1-carboxylic acid tert-butyl ester is replaced with (3 S) - 4-{7-Chloro-6-fluoro-(1 P )-1-[4-methyl-2-(propyl-2-yl)pyridin-3-yl]-2-oxo-1,2- parallel dihydropyrido [2,3- d] pyrimidin-4-yl} -3-methyl-piperazine-1-carboxylic acid tert-butyl ester except using the compound prepared in step 2-1 to give substantially the same (P) -4-((S)-4-Propenyl-2-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (compound 2-2). LCMS: m/z =614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.46 (d, J = 5.0 Hz, 1H), 8.42 – 8.33 (m, 1H), 7.33 – 7.25 (m, 1H), 6.91 – 6.76 (m, 1H) , 6.32 (dd, J = 16.7, 4.7 Hz, 1H), 5.84 (dd, J = 10.6, 1.9 Hz, 1H), 5.28 – 5.08 (m, 1H), 4.63 – 4.36 (m, 2H), 4.24 – 4.03 (m, 1H), 3.96 – 3.79 (m, 1H), 3.79 – 3.54 (m, 1H), 3.45 – 3.35 (m, 0.5H), 3.26 – 3.15 (m, 0.5H), 2.89 – 2.76 (m, 1H), 2.12 – 1.99 (m, 3H), 1.46 (d, J = 6.6 Hz, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.7 Hz, 3H).
實施例 3
4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物3);(M)
-4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮; 和(P)-
4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧代-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)哌嗪-1-甲酸第三丁酯Step 1. 4-(7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridinepara[2, 3-d]pyrimidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester
用氮氣置換500-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入7-氯-6-氟-4-羥基-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (8.24 g, 23.63 mmol)、POCl3 (7.37 g, 48.06 mmol)、DIEA (8.32 g, 64.37 mmol)及乙腈 (80 mL)。混合物70o C攪拌1 h。將反應冷卻至室溫並真空濃縮，得4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其直接用於下一步。A 500-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-methylpyridin-3-yl) pyridin-parallel [2,3-d] pyrimidin -2 (1H) - one (8.24 g, 23.63 mmol), POCl 3 (7.37 g, 48.06 mmol), DIEA (8.32 g, 64.37 mmol) , and acetonitrile (80 mL). 70 o C the mixture was stirred for 1 h. The reaction was cooled to room temperature and concentrated in vacuo to give 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine[2,3-d] Pyrimidine-2(1H)-one, which was used directly in the next step.
用氮氣置換250-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(粗品)及乙腈(80 mL)。加入DIEA (6.01 g, 46.50 mmol)和哌嗪-1-甲酸第三丁酯(544 g, 29.21 mmol)。反應混合物室溫攪拌1 h。然後加水(50 mL)淬滅反應。反應液真空濃縮，殘餘物矽膠柱純化，用EA/正己烷洗脫，得4.38 g (36% 兩步)的4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢 [2,3-d]嘧啶-4-基)哌嗪-1-甲酸第三丁酯，其為黃色固體。LCMS:m/z = 517 [M+1]+ 。A 250-mL round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine Para[2,3-d]pyrimidin-2(1H)-one (crude) and acetonitrile (80 mL). DIEA (6.01 g, 46.50 mmol) and 3-butyl piperazine-1-carboxylate (544 g, 29.21 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. Water (50 mL) was then added to quench the reaction. The reaction solution was concentrated in vacuo, and the residue was purified by silica gel column, eluted with EA/n-hexane to give 4.38 g (36% for two steps) of 4-(7-chloro-6-fluoro-1-(2-isopropyl-4). -Methylpyridin-3-yl)-2-oxo-1,2-dihydropyridinepara[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester as a yellow solid . LCMS: m/z = 517 [M+1] + .
步驟 2. 4-(4-丙烯醯基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 2. 4-(4-Propenylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2 ,3-d]pyrimidin-2(1H)-one
用氮氣置換250-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)哌嗪-1-甲酸第三丁酯 (4.37 g, 8.45 mmol)、TFA (15 ml)和二氯甲烷(40 mL)。反應混合物室溫攪拌0.5 h。將反應混合物真空濃縮，殘餘物用DCM (40 mL)溶於100-mL圓底瓶中，加入TEA (5.30 g, 52.37 mmol)。將反應混合物冷卻至0o C並加入丙烯醯氯 (0.90 g, 9.94 mmol)。混合物室溫攪拌1 h。反應液真空濃縮，殘餘物矽膠柱純化，用EA/hexane (v/v=1/1)洗脫，得1.50 g (23%)的4-(4-丙烯醯基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色固體。LCMS:m/z = 471 [M+1]+ 。A 250-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)- 2-Oxo-1,2-dihydropyridinepara[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (4.37 g, 8.45 mmol), TFA (15 ml) and dimethine Chloromethane (40 mL). The reaction mixture was stirred at room temperature for 0.5 h. The reaction mixture was concentrated in vacuo and the residue was dissolved in DCM (40 mL) in a 100-mL round bottom flask and TEA (5.30 g, 52.37 mmol) was added. The reaction mixture was cooled to 0 o C and adding Bing Xixi chloride (0.90 g, 9.94 mmol). The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated in vacuo, and the residue was purified by silica gel column, eluted with EA/hexane (v/v=1/1), to obtain 1.50 g (23%) of 4-(4-propenylpiperazin-1-yl) -7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as a yellow solid . LCMS: m/z = 471 [M+1] + .
步驟 3. 4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物3)
用氮氣置換40-mL封管並維持其氮氣惰性氛圍，向其中加入4-(4-丙烯醯基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (1.42 g, 3.02 mmol)、2,3,4,5-四氟-6- (4,4,5,5-四甲基-1,3,2-二氧雜環戊硼烷- 2-基)苯胺 (3.32 g, 9.68 mmol)、Pd(PPh3 )4 (0.77 g, 0.67 mmol)、Na2 CO3 (1.04 g, 9.81 mmol)、二氧六環(15 mL)及水(1.5 mL)。反應混合物80o C攪拌1 h。將該反應混合物過濾並真空濃縮。殘餘物用Prep-HPLC純化，用CH3 CN/H2 O(v/v=7/3)洗脫，得535 mg (30 % 收率)的4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物3)，其為黃色固體。LCMS: m/z = 600 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.50 – 8.43 (m, 2H), 7.29 (dd, J = 5.0, 0.8 Hz, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.8, 2.0 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.03 – 3.85 (m, 4H), 2.91 – 2.78 (m, 1H), 2.04 (d, J = 3.6 Hz, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H)。A 40-mL tube was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 4-(4-propenylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl) -4-Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (1.42 g, 3.02 mmol), 2,3,4,5-tetrafluoro-6-( 4,4,5,5-Tetramethyl-1,3,2-dioxaborolane-2-yl)aniline (3.32 g, 9.68 mmol), Pd(PPh 3 ) 4 (0.77 g, 0.67 mmol), Na 2 CO 3 ( 1.04 g, 9.81 mmol), dioxane (15 mL) and water (1.5 mL). 80 o C the reaction mixture was stirred for 1 h. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by Prep-HPLC, eluting with CH 3 CN / H 2 O ( v / v = 7/3), to give 535 mg (30% yield) of 4- (4-Bing Xixi piperazine -1 -yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine Pio[2,3-d]pyrimidin-2(lH)-one (compound 3) as a yellow solid. LCMS: m/z = 600 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.50 – 8.43 (m, 2H), 7.29 (dd, J = 5.0, 0.8 Hz, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.8, 2.0 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.03 – 3.85 (m, 4H), 2.91 – 2.78 (m , 1H), 2.04 (d, J = 3.6 Hz, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H).
步驟 4. 4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4- 甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(第一洗脫異構體，化合物3-1 ) & 4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(第二洗脫異構體，化合物3-2)Step 4. 4-(4-Propenylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2- Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (first eluting isomer, compound 3-1 ) & 4-(4 -Propenylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methyl) pyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second eluting isomer, compound 3-2)
採用以下條件，通過Chiral-Prep-HPLC純化4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(550 mg)的阻轉異構體混合物：柱子：CHIRALPAK IF，2cm × 25cm，5um；流動相：(Hex:DCM=3:1(v/v))：EtOH=90:10(v/v)；檢測波長：UV 220nm，得255 mg(46.36%)的4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物3-1)，其為黃色固體。LCMS：m/z =600 [M+1]+ 。1 H NMR (400 MHz,CD3 OD) δ 8.50 – 8.43 (m, 2H), 7.32 – 7.26 (m, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.8, 2.0 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.03 – 3.86 (m, 4H), 2.93 – 2.78 (m, 1H), 2.04 (s, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H)。 和247 mg (44.91%)的4-(4-丙烯醯基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物3-2)，其為黃色固體。LCMS: m/z =600 [M+1]+ 。1 H NMR (400 MHz,CD3 OD) δ 8.50 – 8.43 (m, 2H), 7.29 (dd, J = 5.0, 0.8 Hz, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.7, 1.9 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.04 – 3.85 (m, 4H), 2.94 – 2.78 (m, 1H), 2.04 (s, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H)。4-(4-Propenylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6 was purified by Chiral-Prep-HPLC using the following conditions - Atropisomer mixture of fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (550 mg) : Column: CHIRALPAK IF, 2cm × 25cm, 5um; Mobile phase: (Hex:DCM=3:1(v/v)): EtOH=90:10(v/v); Detection wavelength: UV 220nm, yield 255 mg (46.36%) 4-(4-Propenylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-( 2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (first eluting isomer, compound 3-1), which is Yellow solid. LCMS: m/z = 600 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.50 – 8.43 (m, 2H), 7.32 – 7.26 (m, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.8, 2.0 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.03 – 3.86 (m, 4H), 2.93 – 2.78 (m, 1H), 2.04 (s, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H). and 247 mg (44.91%) of 4-(4-propenylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6-fluoro- 1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second eluting isomer, compound 3-2) , which is a yellow solid. LCMS: m/z =600 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.50 – 8.43 (m, 2H), 7.29 (dd, J = 5.0, 0.8 Hz, 1H), 6.82 (dd, J = 16.8, 10.6 Hz, 1H), 6.30 (dd, J = 16.7, 1.9 Hz, 1H), 5.83 (dd, J = 10.6, 2.0 Hz, 1H), 4.26 – 4.09 (m, 4H), 4.04 – 3.85 (m, 4H), 2.94 – 2.78 (m , 1H), 2.04 (s, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H).
實施例4
4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物4);(M)
-4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮;(P)
-4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 2,2,2-三氟-N-(2,3,4,5-四氟苯基)乙醯胺Step 1. 2,2,2-Trifluoro-N-(2,3,4,5-tetrafluorophenyl)acetamide
用氮氣置換1000-mL三口圓底燒瓶並維持其氮氣惰性氛圍，向其中加入2,3,4,5-四氟苯胺 (29.64 g, 179.54 mmol)、TEA (49.02 g,484.43 mmol)及DCM (300 mL)並攪拌。將混合物冷卻至0o C，然後加入三氟乙酸酐(61.67 g, 293.62 mmol)。所得溶液室溫進一步攪拌2 h。反應液真空濃縮，粗品加水(500 mL)淬滅，所得溶液用EA (3 x 200 mL)萃取，合併有機相，然後用碳酸氫鈉水溶液 (200 mL)和飽和食鹽水(300 mL)洗滌，無水Na2 SO4 乾燥，殘餘物真空濃縮得53.45 g (粗品)的2,2,2-三氟-N-(2,3,4,5-四氟苯基)乙醯胺。LCMS：m/z = 260 [M-H]- 。A 1000-mL three-necked round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which were added 2,3,4,5-tetrafluoroaniline (29.64 g, 179.54 mmol), TEA (49.02 g, 484.43 mmol) and DCM ( 300 mL) and stirred. The mixture was cooled to 0 o C, followed by addition of trifluoroacetic anhydride (61.67 g, 293.62 mmol). The resulting solution was further stirred at room temperature for 2 h. The reaction solution was concentrated in vacuo, the crude product was quenched by adding water (500 mL), the resulting solution was extracted with EA (3 x 200 mL), the organic phases were combined, and then washed with aqueous sodium bicarbonate solution (200 mL) and saturated brine (300 mL), Dry over anhydrous Na 2 SO 4 and concentrate the residue in vacuo to give 53.45 g (crude) of 2,2,2-trifluoro-N-(2,3,4,5-tetrafluorophenyl)acetamide. LCMS: m/z = 260 [MH] - .
步驟 2. (2-胺基-3,4,5,6-四氟苯基)硼酸Step 2. (2-Amino-3,4,5,6-tetrafluorophenyl)boronic acid
用氮氣置換1000-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入2,2,2-三氟-N-(2,3,4,5-四氟苯基)乙醯胺 (25.03 g, 95.90 mmol)和THF (250 mL)。混合物降至-65o C，然後加入正丁基鋰溶液(88 mL, 223.46 mmol)。混合物-50o C攪拌1 h，然後-70o C滴加硼酸三甲酯(30.24 g, 290.95 mmol)。混合物室溫攪拌1 h，然後加入4N HCl溶液(200 mL)淬滅反應。混合物室溫攪拌0.5 h。所得溶液用EA (2 x 200 mL)萃取，合併有機相，然後用碳酸氫鈉水溶液(200 mL)和飽和食鹽水(200 mL)洗滌，無水Na2 SO4 乾燥，殘餘物真空濃縮。將該粗品於Hex/EA(v/v=20/1)(70 mL)中攪拌1 h。所得固體過濾得5.10 g (25.50% 產率)的(2-胺基-3,4,5,6-四氟苯基)硼酸，其為類白色固體。LCMS：m/z = 210 [M+1]+ 。A 1000-mL round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 2,2,2-trifluoro-N-(2,3,4,5-tetrafluorophenyl)acetamide (25.03 g , 95.90 mmol) and THF (250 mL). The mixture was reduced to -65 o C, then n-butyllithium solution (88 mL, 223.46 mmol) was added. -50 o C the mixture was stirred for 1 h, then -70 o C was added dropwise trimethyl borate (30.24 g, 290.95 mmol). The mixture was stirred at room temperature for 1 h, then 4N HCl solution (200 mL) was added to quench the reaction. The mixture was stirred at room temperature for 0.5 h. The resulting solution was extracted with EA (2 x 200 mL), and the organic phases were combined, washed with aqueous sodium bicarbonate solution (200 mL) and saturated brine (200 mL), dried over anhydrous Na 2 SO 4 , and the residue was concentrated in vacuo. The crude product was stirred in Hex/EA (v/v=20/1) (70 mL) for 1 h. The resulting solid was filtered to give 5.10 g (25.50% yield) of (2-amino-3,4,5,6-tetrafluorophenyl)boronic acid as an off-white solid. LCMS: m/z = 210 [M+1] + .
步驟 3. 2,3,4,5-四氟-6-((3aR,4R,6R)-3a,5,5-三甲基六氫-4,6-甲氧基苯[d][1,3,2]二氧雜硼-基)苯胺
向250-mL圓底燒中加入(2-胺基-3,4,5,6-四氟苯基)硼酸(11.82 g，56.58 mmol)、(1S,3R,4S,5S)-4,6,6-三甲基雙環[3.1.1]庚烷-3,4-二醇 (9.56 g, 56.58 mmol)和甲苯(120 mL)。混合物55o C攪拌3h，殘餘物真空濃縮並用矽膠柱純化，用Hex/EA(v/v=20/1)洗脫，得17.08 g (87.98%產率)的2,3,4,5-四氟-6-((3aR,4R,6R)-3a,5,5-三甲基六氫-4,6-甲氧基苯[d][1,3,2]二氧雜硼-2-基)苯胺，其為棕色固體。LCMS:m/z = 344 [M+1]+ 。To a 250-mL round-bottomed flask was added (2-amino-3,4,5,6-tetrafluorophenyl)boronic acid (11.82 g, 56.58 mmol), (1S,3R,4S,5S)-4,6 ,6-trimethylbicyclo[3.1.1]heptane-3,4-diol (9.56 g, 56.58 mmol) and toluene (120 mL). The mixture was stirred at 55 o C for 3 h, the residue was concentrated in vacuo and purified with a silica gel column, eluted with Hex/EA (v/v=20/1) to give 17.08 g (87.98% yield) of 2,3,4,5- Tetrafluoro-6-((3aR,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methoxybenzene[d][1,3,2]dioxaborol-2 -yl)aniline as a brown solid. LCMS: m/z = 344 [M+1] + .
步驟 4. (R)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-2-甲基哌嗪-1-甲酸第三丁酯Step 4. (R)-4-(7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine [2,3-d]pyrimidin-4-yl)-2-methylpiperazine-1-carboxylic acid tert-butyl ester
用氮氣置換50-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2,4(1H,3H)-二酮(7.24 g, 0.02 mol)、POCl3 (7.95 g, 0.05 mol)、DIEA (12.12 g, 0.12 mol)和乙腈(30 mL)。混合物80o C攪拌2 h。將反應冷卻至室溫並真空濃縮，得4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其直接用於下一步。A 50-mL round-bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridin[2 , 3-d] pyrimidine -2,4 (1H, 3H) - dione (7.24 g, 0.02 mol), POCl 3 (7.95 g, 0.05 mol), DIEA (12.12 g, 0.12 mol) and acetonitrile (30 mL) . 80 o C the mixture was stirred for 2 h. The reaction was cooled to room temperature and concentrated in vacuo to give 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine[2,3-d] Pyrimidine-2(1H)-one, which was used directly in the next step.
用氮氣置換100-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(粗品)和乙腈(80 mL)，隨後加入DIEA (12.12 g, 0.12 mol)和(R)-2-甲基哌嗪-1-甲酸第三丁酯(4.01 g, 0.02 mol)。反應混合物室溫攪拌0.5 h，然後加水(300 mL)淬滅反應。所得溶液用乙酸乙酯 (2 x 300 mL)萃取，合併有機相並用飽和食鹽水洗滌(50 mL)，無水Na2 SO4 乾燥，過濾並真空濃縮。所得殘餘物矽膠柱純化，用EA/正己烷(v:v=9/1)洗脫，得3.208 g (30% 兩步)的(R)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-2-甲基哌嗪-1-甲酸第三丁酯，其為黃色固體。LCMS:m/z = 531 [M+1]+ 。A 100-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine Para[2,3-d]pyrimidin-2(1H)-one (crude) and acetonitrile (80 mL), followed by DIEA (12.12 g, 0.12 mol) and (R)-2-methylpiperazine-1- 3-Butyl formate (4.01 g, 0.02 mol). The reaction mixture was stirred at room temperature for 0.5 h, then water (300 mL) was added to quench the reaction. The resulting solution was extracted with ethyl acetate (2 x 300 mL), the organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The resulting residue was purified by silica gel column eluting with EA/n-hexane (v:v=9/1) to give 3.208 g (30% for two steps) of (R)-4-(7-chloro-6-fluoro-1) -(2-Isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-2-methylpiperidine tert-butyl oxazine-1-carboxylate as a yellow solid. LCMS: m/z = 531 [M+1] + .
步驟 5. (R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 5. (R)-4-(4-Propenyl-3-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridine -3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
用氮氣置換50-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入(R)-4-(7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-2-氧-1,2-二氫吡啶駢[2,3-d]嘧啶-4-基)-2-甲基哌嗪-1-甲酸第三丁酯 (3.208 g, 6.04 mol)、TFA(25 ml)和DCM (75 mL)。反應混合物室溫攪拌1 h並真空濃縮，得(R)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-4-(3-甲基哌嗪-1-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為粗品，直接用於下一步反應。A 50-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (R)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridine-3) -yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-4-yl)-2-methylpiperazine-1-carboxylic acid tert-butyl ester (3.208 g, 6.04 mol ), TFA (25 ml) and DCM (75 mL). The reaction mixture was stirred at room temperature for 1 h and concentrated in vacuo to give (R)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-4-(3-methyl) ylpiperazin-1-yl)pyridino[2,3-d]pyrimidin-2(1H)-one, which was crude and used directly in the next reaction.
用氮氣置換50-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入(R)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)-4-(3-甲基哌嗪-1-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (粗品)、ACN(75 mL)、DIEA (1.45 g, 12.08 mmol)和丙烯醯氯(0.549 g, 6.06 mmol)。反應混合物室溫攪拌0.5h，然後加水(100 mL)淬滅反應，用乙酸乙酯(3 x200 mL)萃取。合併有機相並用飽和食鹽水(100 mL)洗滌，無水Na2 SO4 乾燥，過濾並真空濃縮，殘餘物矽膠柱純化，用EA/正己烷(v:v=4/1)洗脫，得2.38 g (82% 兩步)的(R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色固體。LCMS:m/z = 485[M+1]+ 。A 50-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (R)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl) -4-(3-Methylpiperazin-1-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (crude), ACN (75 mL), DIEA (1.45 g, 12.08 mmol) and allyl chloride (0.549 g, 6.06 mmol). The reaction mixture was stirred at room temperature for 0.5 h, then quenched with water (100 mL) and extracted with ethyl acetate (3 x 200 mL). The organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column and eluted with EA/n-hexane (v:v=4/1) to obtain 2.38 g (82% two steps) of (R)-4-(4-propenyl-3-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl- 4-Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as a yellow solid. LCMS: m/z = 485[M+1] + .
步驟 6. 4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟- 1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物4)Step 6. 4-((R)-4-Propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)- 6-Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidin-2(1H)-one (Compound 4)
用氮氣置換20-mL封管並維持其氮氣惰性氛圍，向其中加入(R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(1.224 g, 2.52 mmol)、2,3,4,5-四氟-6-((3aR,4R,6R)-3a,5,5-三甲基六氫-4,6-甲氧基苯[d][1,3,2]二氧雜硼-2-基)苯胺(1.794 g, 5.223 mmol)、Pd(PPh3 )4 (0.614 g, 0.53 mmol)、Na2 CO3 (0.72g, 6.79 mmol)、二氧六環(12 mL)及水(1 mL)。反應混合物80o C攪拌2 h。反應混合物過濾並真空濃縮，殘餘物通過Prep-HPLC純化，用ACN/H2 O (1%NH4 HCO3 ) (v:v=7/3)洗脫，得740 mg (47.8% 兩步)的4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物4)，其為黃色固體。LCMS:m/z = 614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.52 – 8.43 (m, 2H), 7.29 (d,J = 5.0 Hz, 1H), 6.81 (dd,J = 16.8, 10.6 Hz, 1H), 6.29 (d,J = 16.5 Hz, 1H), 5.81 (d,J = 10.4 Hz, 1H), 4.58 – 4.26 (m, 3H), 4.11 – 3.72 (m, 4H), 2.81– 2.83 (m, 1H), 2.05 (d,J = 18.6 Hz, 3H), 1.39 (s, 3H), 1.18 (d,J = 6.8 Hz, 3H), 1.01– 1.02 (m, 3H)。A 20-mL tube was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (R)-4-(4-propenyl-3-methylpiperazin-1-yl)-7-chloro-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (1.224 g, 2.52 mmol), 2,3,4 ,5-Tetrafluoro-6-((3aR,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methoxybenzene[d][1,3,2]dioxa boron-2-yl) aniline (1.794 g, 5.223 mmol), Pd (PPh 3) 4 (0.614 g, 0.53 mmol), Na 2 CO 3 (0.72g, 6.79 mmol), dioxane (12 mL) and water (1 mL). 80 o C the reaction mixture was stirred for 2 h. The reaction mixture was filtered and concentrated in vacuo, the residue was purified by Prep-HPLC, ACN / H 2 O (1% NH 4 HCO 3) with (v: v = 7/3 ) as eluent to give 740 mg (47.8% two steps) 4-((R)-4-Propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)-6- Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 4) as a yellow solid. LCMS: m/z = 614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.52 – 8.43 (m, 2H), 7.29 (d, J = 5.0 Hz, 1H), 6.81 (dd, J = 16.8, 10.6 Hz, 1H), 6.29 (d , J = 16.5 Hz, 1H), 5.81 (d, J = 10.4 Hz, 1H), 4.58 – 4.26 (m, 3H), 4.11 – 3.72 (m, 4H), 2.81 – 2.83 (m, 1H), 2.05 ( d, J = 18.6 Hz, 3H), 1.39 (s, 3H), 1.18 (d, J = 6.8 Hz, 3H), 1.01– 1.02 (m, 3H).
步驟 7. 4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物4-1 ) & 4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物4-2)Step 7. 4-((R)-4-Propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl)- 6-Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (first eluting isomer, compound 4-1 ) & 4-((R)-4-propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl )-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second eluting isomer , compound 4-2)
採用以下條件，通過Chiral-Prep-HPLC純化4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(710 mg)的阻轉異構體混合物：柱子：CHIRAL ART Amylose-SA，3cm × 25cm，5um；流動相：CO2 : IPA(v/v =62:38)；檢測波長：UV 220nm，得223 mg (40.58%)的4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(第一洗脫異構體，化合物4-1)，其為黃色固體。LCMS:m/z =614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.46 (dd,J = 7.3, 5.0 Hz, 2H), 7.34 – 7.21 (m, 1H), 6.81 (dd,J = 16.7, 10.6 Hz, 1H), 6.29 (d,J = 16.7 Hz, 1H), 5.81 (d,J = 11.0 Hz, 1H), 4.58 – 4.32 (m, 3H), 3.98 – 3.77 (m, 4H), 2.75 – 2.77 (m, 1H), 2.07 (s, 3H), 1.50 – 1.30 (m, 3H), 1.18 (d,J = 6.8 Hz, 3H), 1.00 (d,J = 6.8 Hz, 3H)。 和236 mg(38.84%)的4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物4-2)，其為黃色固體。LCMS:m/z =614 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.50 (d,J = 8.9 Hz, 1H), 8.40 (d,J = 5.0 Hz, 1H), 7.24 (d,J = 4.5 Hz, 1H), 6.81 (dd,J = 16.9, 10.4 Hz, 1H), 6.29 (d,J = 16.9 Hz, 1H), 5.81 (d,J = 10.5 Hz, 1H), 4.40 – 4.42 (m, 3H), 4.08 – 3.80 (m, 4H), 2.87 – 2.69 (m, 1H), 2.02 (s, 3H), 1.48 – 1.32 (m, 3H), 1.20 – 1.16 (m, 3H), 1.00 (d,J = 6.8 Hz, 3H)。4-((R)-4-Propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5, 6-Tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (710 mg) of atropisomeric mixture: column: CHIRAL ART Amylose-SA, 3cm × 25cm, 5um; mobile phase: CO 2 : IPA (v/v =62:38); detection wavelength: UV 220nm, yield 223 mg (40.58%) 4-((R)-4-propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluorophenyl) )-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (first eluting isomer , compound 4-1), which is a yellow solid. LCMS: m/z =614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.46 (dd, J = 7.3, 5.0 Hz, 2H), 7.34 – 7.21 (m, 1H), 6.81 (dd, J = 16.7, 10.6 Hz, 1H), 6.29 (d, J = 16.7 Hz, 1H), 5.81 (d, J = 11.0 Hz, 1H), 4.58 – 4.32 (m, 3H), 3.98 – 3.77 (m, 4H), 2.75 – 2.77 (m, 1H), 2.07 (s, 3H), 1.50 – 1.30 (m, 3H), 1.18 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H). and 236 mg (38.84%) of 4-((R)-4-propenyl-3-methylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetra Fluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (second elution isomer, compound 4-2) as a yellow solid. LCMS: m/z =614 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.50 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.24 (d, J = 4.5 Hz, 1H), 6.81 ( dd, J = 16.9, 10.4 Hz, 1H), 6.29 (d, J = 16.9 Hz, 1H), 5.81 (d, J = 10.5 Hz, 1H), 4.40 – 4.42 (m, 3H), 4.08 – 3.80 (m , 4H), 2.87 – 2.69 (m, 1H), 2.02 (s, 3H), 1.48 – 1.32 (m, 3H), 1.20 – 1.16 (m, 3H), 1.00 (d, J = 6.8 Hz, 3H).
實施例 5
4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物5);(M)
-4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮;和(P)
-4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 1. 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl- 4-Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
用氮氣置換250-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入7-氯-6-氟-4-羥基-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (7.75 g, 22.22 mmol)、POCl3 (8.75 g, 57.08 mmol)、DIEA(10.00g, 77.35 mmol)及乙腈(80 mL)。混合物80o C攪拌1 h。將反應冷卻至室溫並真空濃縮。用氮氣置換250-mL圓底燒瓶並維持其氮氣氛圍，向其中加入4,7-二氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (8.17 g, 22.22 mmol)和乙腈(80 mL)。加入DIEA (3.24 g, 25.09 mmol)和(2R,6S)-2,6-二甲基哌嗪 (2.36 g, 20.69 mmol)，反應混合物室溫攪拌0.5 h。將反應混合物冷卻至0o C並加入丙烯醯氯(1.65 g, 18.21 mmol)，然後加水(50 mL)淬滅反應，所得溶液用乙酸乙酯(3 x 150 mL)萃取，合併有機相並用飽和食鹽水洗滌(50 mL)，無水Na2 SO4 乾燥，過濾並真空濃縮。殘餘物矽膠柱純化，用MeOH/EA(v/v=1/10)洗脫，得3.70 g (33%兩步)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色油。LCMS:m/z = 499 [M+1]+ 。A 250-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-methylpyridin-3-yl) pyridin-parallel [2,3-d] pyrimidin -2 (1H) - one (7.75 g, 22.22 mmol), POCl 3 (8.75 g, 57.08 mmol), DIEA (10.00g, 77.35 mmol) , and acetonitrile (80 mL). 80 o C the mixture was stirred for 1 h. The reaction was cooled to room temperature and concentrated in vacuo. A 250-mL round bottom flask was replaced with nitrogen and maintained under nitrogen, to which was added 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine [2,3-d]pyrimidin-2(1H)-one (8.17 g, 22.22 mmol) and acetonitrile (80 mL). DIEA (3.24 g, 25.09 mmol) and (2R,6S)-2,6-dimethylpiperazine (2.36 g, 20.69 mmol) were added and the reaction mixture was stirred at room temperature for 0.5 h. The reaction mixture was cooled to 0 o C and adding Bing Xixi chloride (1.65 g, 18.21 mmol), then water (50 mL) quenched the reaction, (3 x 150 mL) The resulting solution was extracted with ethyl acetate, the organic phases were combined and washed with saturated Washed with brine (50 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column eluting with MeOH/EA (v/v=1/10) to give 3.70 g (33% for two steps) of 4-((3S,5R)-4-propenyl-3,5 -Dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine- 2(1H)-ketone as a yellow oil. LCMS: m/z = 499 [M+1] + .
步驟 2. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物5)Step 2. 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrakis Fluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 5)
向20-mL圓底燒瓶中加入4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(0.295 g, 0.59 mmol)、(2-胺基-3,4,5,6-四氟苯基)硼酸(0.348 g, 1.67 mmol)、Pd(PPh3 )4 (0.140 g, 0.12 mmol)、Na2 CO3 (0.214 g, 2.02 mmol)、二氧六環 (5 mL)和水(1 mL)。反應混合物80o C攪拌1 h。將反應混合物過濾並真空濃縮，殘餘物通過Prep-HPLC純化，使用CH3 CN/H2 O(0.05%NH4 HCO3 )(v:v=2/1)洗脫，得0.111 g (30%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物5)，其為黃色固體. LCMS:m/z =628 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.39 – 8.32 (m, 2H), 7.21 (d,J = 5.0 Hz, 1H), 6.77 – 6.70 (m, 1H), 6.24 – 6.18 (m, 1H), 5.78 – 5.69 (m, 1H), 4.60 (s, 2H), 4.46 – 4.35 (m, 2H), 3.77 – 3.67 (m, 2H), 2.74 – 2.66 (m, 1H), 1.97 (s, 3H), 1.43 (d,J = 6.9 Hz, 3H), 1.37 (d,J = 7.0 Hz, 3H), 1.09 (d,J = 6.8 Hz, 3H), 0.93 (d,J = 6.8 Hz, 3H)。To a 20-mL round bottom flask was added 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-( 2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (0.295 g, 0.59 mmol), (2-amino-3,4 ,5,6-tetrafluorophenyl)boronic acid (0.348 g, 1.67 mmol), Pd(PPh 3 ) 4 (0.140 g, 0.12 mmol), Na 2 CO 3 (0.214 g, 2.02 mmol), dioxane ( 5 mL) and water (1 mL). 80 o C the reaction mixture was stirred for 1 h. The reaction mixture was filtered and concentrated in vacuo, the residue was purified by Prep-HPLC, using CH 3 CN / H 2 O ( 0.05% NH 4 HCO 3) (v: v = 2/1) as eluent to give 0.111 g (30% ) 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(2-amino-3,4,5,6-tetrafluoro Phenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Compound 5), which As a yellow solid. LCMS: m/z =628 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.39 – 8.32 (m, 2H), 7.21 (d, J = 5.0 Hz, 1H), 6.77 – 6.70 (m, 1H), 6.24 – 6.18 (m, 1H) , 5.78 – 5.69 (m, 1H), 4.60 (s, 2H), 4.46 – 4.35 (m, 2H), 3.77 – 3.67 (m, 2H), 2.74 – 2.66 (m, 1H), 1.97 (s, 3H) , 1.43 (d, J = 6.9 Hz, 3H), 1.37 (d, J = 7.0 Hz, 3H), 1.09 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H).
步驟 3. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(第一洗脫異構體，化合物5-1) & 4-((3S,5R)-4-丙烯醯基-3,5- 二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(第二洗脫異構體，化合物5-2)
採用以下條件，通過Chiral-Prep-HPLC純化4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟 苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(408 mg)的阻轉異構體混合物：柱子：CHIRALPAK IG，3cm × 25cm，5um；流動相：(Hex:DCM=3:1(v/v))：EtOH=92:8(v/v)；檢測波長：UV 220nm，得148 mg (36.27%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物5-1)，其為黃色固體。LCMS:m/z =628 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.39 – 8.31 (m, 2H), 7.20 (d,J = 5.0 Hz, 1H), 6.79 – 6.68 (m, 1H), 6.24 – 6.16 (m, 1H), 5.75 – 5.68 (m, 1H), 4.56 (d,J = 40.4 Hz, 2H), 4.48 – 4.26 (m, 2H), 3.89 – 3.61 (m, 2H), 2.76 – 2.64 (m, 1H), 1.96 (s, 3H), 1.54 – 1.28 (m, 6H), 1.09 (d,J = 6.8 Hz, 3H), 0.97 – 0.81 (m, 3H)。 和159 mg (38.97%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(2-胺基-3,4,5,6-四氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物5-2)，其為黃色固體。LCMS:m/z =628 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.40 – 8.30 (m, 2H), 7.20 (d,J = 5.0 Hz, 1H), 6.78 – 6.67 (m, 1H), 6.25 – 6.16 (m, 1H), 5.78 – 5.67 (m, 1H), 4.56 (d,J = 38.1 Hz, 2H), 4.51 – 4.33 (m, 2H), 3.83 – 3.66 (m, 2H), 2.78 – 2.70 (m, 1H), 1.96 (s, 3H), 1.48 – 1.38 (m, 3H), 1.36 (d,J = 6.9 Hz, 3H), 1.09 (d,J = 6.8 Hz, 3H), 0.92 (d,J = 6.8 Hz, 3H)。4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(2-amino-3 was purified by Chiral-Prep-HPLC using the following conditions ,4,5,6-Tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine-2(1H )-ketone (408 mg) atropisomer mixture: Column: CHIRALPAK IG, 3cm × 25cm, 5um; Mobile phase: (Hex:DCM=3:1(v/v)):EtOH=92:8( v/v); detection wavelength: UV 220nm, obtained 148 mg (36.27%) of 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7 -(2-Amino-3,4,5,6-tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3 -d]pyrimidin-2(1H)-one (first eluting isomer, compound 5-1) as a yellow solid. LCMS: m/z =628 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.39 – 8.31 (m, 2H), 7.20 (d, J = 5.0 Hz, 1H), 6.79 – 6.68 (m, 1H), 6.24 – 6.16 (m, 1H) , 5.75 – 5.68 (m, 1H), 4.56 (d, J = 40.4 Hz, 2H), 4.48 – 4.26 (m, 2H), 3.89 – 3.61 (m, 2H), 2.76 – 2.64 (m, 1H), 1.96 (s, 3H), 1.54 – 1.28 (m, 6H), 1.09 (d, J = 6.8 Hz, 3H), 0.97 – 0.81 (m, 3H). and 159 mg (38.97%) of 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(2-amino-3,4, 5,6-Tetrafluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (Second eluting isomer, compound 5-2) as a yellow solid. LCMS: m/z =628 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.40 – 8.30 (m, 2H), 7.20 (d, J = 5.0 Hz, 1H), 6.78 – 6.67 (m, 1H), 6.25 – 6.16 (m, 1H) , 5.78 – 5.67 (m, 1H), 4.56 (d, J = 38.1 Hz, 2H), 4.51 – 4.33 (m, 2H), 3.83 – 3.66 (m, 2H), 2.78 – 2.70 (m, 1H), 1.96 (s, 3H), 1.48 – 1.38 (m, 3H), 1.36 (d, J = 6.9 Hz, 3H), 1.09 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H) .
實施例6
4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物6)
步驟 1. (R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 1. (R)-4-(4-Propenyl-3-methylpiperazin-1-yl)-7-(5-amino-2,3,4-trifluorophenyl)-6- Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
用氮氣置換20-mL密封管並維持其氮氣氛圍，向其中加入(R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮(1.02 g, 2.47 mmol)、(5-胺基-2,3,4-三氟苯基)硼酸(0.94 g, 4.92 mmol)、Pd(PPh3 )4 (0.59 g, 0.51 mmol)、Na2 CO3 (0.738 g, 6.96 mmol)、二氧六環 (30 mL)和水(2 mL)。反應混合物80o C攪拌4 h。將該反應混合物過濾並真空濃縮，殘餘物用矽膠柱純化，用ACN/H2 O(v/v=7/3)洗脫，得200 mg (粗品)的(R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色固體。LCMS:m/z = 596 [M+1]+ 。A 20-mL sealed tube was replaced with nitrogen and maintained under nitrogen, to which was added (R)-4-(4-propenyl-3-methylpiperazin-1-yl)-7-chloro-6-fluoro- 1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (1.02 g, 2.47 mmol), (5-amino- 2,3,4-trifluorophenyl) borate (0.94 g, 4.92 mmol), Pd (PPh 3) 4 (0.59 g, 0.51 mmol), Na 2 CO 3 (0.738 g, 6.96 mmol), dioxane (30 mL) and water (2 mL). 80 o C the reaction mixture was stirred for 4 h. The reaction mixture was filtered and concentrated in vacuo, the residue was purified by silica gel column, eluting with ACN / H 2 O (v / v = 7/3), to give 200 mg (crude) of (R) -4- (4- Acryloyl-3-methylpiperazin-1-yl)-7-(5-amino-2,3,4-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4 -Methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as a yellow solid. LCMS: m/z = 596 [M+1] + .
步驟 2. 4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物6)Step 2. 4-((R)-4-Propenyl-3-methylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl )-6-Fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidin-2(1H)-one (Compound 6)
用氮氣置換50-mL圓底燒瓶並維持其氮氣惰性氛圍，向其中加入(R)-4-(4-丙烯醯基-3-甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (200 mg, 0.34 mmol)、NCS (111 mg, 0.83 mmol)及冰醋酸 (3 mL)。反應混合物室溫攪拌48 h，然後加水(100 mL)淬滅該反應。所得溶液用乙酸乙酯 (2 x 100 mL)萃取，合併有機相並用飽和食鹽水洗滌(10 mL)，無水Na2 SO4 乾燥，過濾並真空濃縮，殘餘物通過Prep-HPLC純化，用ACN/H2 O(0.5%NH4 HCO3 )(v/v =7/3)洗脫，得17 mg的4-((R)-4-丙烯醯基-3-甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物6)，其為黃色固體。LCMS:m/z = 630[M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.50 (d,J = 8.9 Hz, 1H), 8.40 (d,J = 5.0 Hz, 1H), 7.24 (d,J = 4.5 Hz, 1H), 6.81 (dd,J = 16.9, 10.4 Hz, 1H), 6.29 (d,J = 16.9 Hz, 1H), 5.81 (d,J = 10.5 Hz, 1H), 4.40– 4.41 (m, 3H), 4.15 – 3.55 (m, 4H), 2.87 – 2.69 (m, 1H), 2.07– 1.94 (m, 3H), 1.49– 1.33 (m, 3H), 1.20 – 1.16 (m, 3H), 1.01– 1.03 (m, 3H)。A 50-mL round bottom flask was replaced with nitrogen and maintained in an inert atmosphere of nitrogen, to which was added (R)-4-(4-propenyl-3-methylpiperazin-1-yl)-7-(5-amine ( 1H)-ketone (200 mg, 0.34 mmol), NCS (111 mg, 0.83 mmol) and glacial acetic acid (3 mL). The reaction mixture was stirred at room temperature for 48 h, then water (100 mL) was added to quench the reaction. The resulting solution was extracted with ethyl acetate (2 x 100 mL), the organic phases were combined and washed with saturated brine (10 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo, the residue was purified by Prep-HPLC with ACN/ Elution with H 2 O (0.5% NH 4 HCO 3 ) (v/v = 7/3) gave 17 mg of 4-((R)-4-propenyl-3-methylpiperazin-1-yl )-7-(3-Amino-2-chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine Para[2,3-d]pyrimidin-2(1H)-one (Compound 6) as a yellow solid. LCMS: m/z = 630[M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.50 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.24 (d, J = 4.5 Hz, 1H), 6.81 ( dd, J = 16.9, 10.4 Hz, 1H), 6.29 (d, J = 16.9 Hz, 1H), 5.81 (d, J = 10.5 Hz, 1H), 4.40– 4.41 (m, 3H), 4.15 – 3.55 (m , 4H), 2.87 – 2.69 (m, 1H), 2.07– 1.94 (m, 3H), 1.49– 1.33 (m, 3H), 1.20 – 1.16 (m, 3H), 1.01– 1.03 (m, 3H).
實施例7
4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (化合物7);(M)
-4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮;和(P)
-4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮
步驟 1. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 1. 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(5-amino-2,3,4-trifluorobenzene yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
向20-mL圓底燒瓶中加入4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-氯-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (2.50 g, 5.01 mmol)、(5-胺基-2,3,4-三氟苯基)硼酸 (3.08 g, 16.15 mmol)、Pd(PPh3
)4
(0.45 g, 0.39 mmol)、Na2
CO3
(0.98 g, 9.21 mmol)、二氧六環(20 mL)及水(4 mL)。反應混合物80o
C攪拌1 h。將該反應混合物過濾並真空濃縮。殘餘物通過C18
柱子純化，使用CH3
CN/H2
O(v/v=2/1)洗脫，得0.476 g的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為黃色固體。LCMS:m/z
=610 [M+1]+
。To a 20-mL round bottom flask was added 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-( 2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (2.50 g, 5.01 mmol), (5-amino-2,3 ,4-trifluorophenyl)boronic acid (3.08 g, 16.15 mmol), Pd(PPh 3 ) 4 (0.45 g, 0.39 mmol), Na 2 CO 3 (0.98 g, 9.21 mmol), dioxane (20 mL) ) and water (4 mL). 80 o C the reaction mixture was stirred for 1 h. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by C 18 column eluting with CH 3 CN / H 2 O elution (v / v = 2/1 ), to give 0.476 g of 4 - ((3S, 5R) -4-
步驟 2. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟 苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮Step 2. 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6 -Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one
向20-mL圓底燒瓶氮氣中加入4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(5-胺基-2,3,4-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (0.476 g, 0.78 mmol)、NCS(0.212 g, 1.59 mmol)及冰醋酸 (5 mL)。反應混合物室溫攪拌1天，將該反應混合物過濾並真空濃縮。殘餘物通過C18 純化，用CH3 CN/H2 O(0.05%NH4 HCO3 )(v/v=1/1)洗脫，得181 mg (37%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮，其為類白色固體。LCMS:m/z =644 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.47 (d,J = 8.9 Hz, 1H), 8.40 (d,J = 5.0 Hz, 1H), 7.27 – 7.22 (m, 1H), 6.92 – 6.77 (m, 1H), 6.37 – 6.22 (m, 1H), 5.84 – 5.75 (m, 1H), 4.70 (s, 2H), 4.60 – 4.40 (m, 2H), 3.91 – 3.71 (m, 2H), 2.87 – 2.74 (m, 1H), 2.11 – 1.99 (m, 3H), 1.60 – 1.37 (m, 6H), 1.18 (d,J = 6.8 Hz, 3H), 1.04 – 0.93 (m, 3H)。To a 20-mL round-bottom flask nitrogen was added 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(5-amino-2, 3,4-Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one (0.476 g, 0.78 mmol), NCS (0.212 g, 1.59 mmol) and glacial acetic acid (5 mL). The reaction mixture was stirred at room temperature for 1 day, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by 18 C, eluting with CH 3 CN / H 2 O ( 0.05% NH 4 HCO 3) (v / v = 1/1), to give 181 mg (37%) of 4 - ((3S, 5R )-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(3-amino-2-chloro-4,5,6-trifluorophenyl)-6-fluoro -1-(2-Isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidin-2(1H)-one as an off-white solid. LCMS: m/z =644 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.47 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.27 – 7.22 (m, 1H), 6.92 – 6.77 (m , 1H), 6.37 – 6.22 (m, 1H), 5.84 – 5.75 (m, 1H), 4.70 (s, 2H), 4.60 – 4.40 (m, 2H), 3.91 – 3.71 (m, 2H), 2.87 – 2.74 (m, 1H), 2.11 – 1.99 (m, 3H), 1.60 – 1.37 (m, 6H), 1.18 (d, J = 6.8 Hz, 3H), 1.04 – 0.93 (m, 3H).
步驟 3. 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟 苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體, 化合物7-1 ) & 4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體, 化合物7-2)
採用以下條件，通過Chiral-Prep-HPLC純化4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟 苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (180 mg)的阻轉異構體混合物：柱子：CHIRALPAK IF, 2cm × 25cm，5um；流動相：(Hex:DCM(v/v =3:1)):IPA(v/v =90:10)；檢測波長：UV 220nm，得81 mg(45%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第一洗脫異構體，化合物7-1)，其為類白色固體。LCMS:m/z =644 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.47 (d,J = 8.9 Hz, 1H), 8.40 (d,J = 5.0 Hz, 1H), 7.31 – 7.20 (m, 1H), 6.86 – 6.77 (m, 1H), 6.34 – 6.25 (m, 1H), 5.87 – 5.76 (m, 1H), 4.81 – 4.62 (m, 2H), 4.62 – 4.39 (m, 2H), 3.92 – 3.75 (m, 2H), 2.85 – 2.72 (m, 1H), 2.03 (d,J = 10.7 Hz, 3H), 1.56 – 1.39 (m, 6H), 1.18 (d,J = 6.8 Hz, 3H), 1.04 – 0.95 (m, 3H)。 和79 mg (43.89%)的4-((3S,5R)-4-丙烯醯基-3,5-二甲基哌嗪-1-基)-7-(3-胺基-2-氯-4,5,6-三氟苯基)-6-氟-1-(2-異丙基-4-甲基吡啶-3-基)吡啶駢[2,3-d]嘧啶-2(1H)-酮 (第二洗脫異構體，化合物7-2)，其為類白色固體。LCMS:m/z =644 [M+1]+ 。1 H NMR (400 MHz, CD3 OD) δ 8.47 (d,J = 8.9 Hz, 1H), 8.40 (d,J = 5.0 Hz, 1H), 7.32 – 7.18 (m, 1H), 6.86 – 6.75 (m, 1H), 6.32 – 6.22 (m, 1H), 5.86 – 5.75 (m, 1H), 4.70 (s, 2H), 4.60 – 4.38 (m, 2H), 3.87 – 3.76 (m, 2H), 2.86 – 2.71 (m, 1H), 2.03 (d,J = 10.7 Hz, 3H), 1.60 – 1.42 (m, 6H), 1.18 (d,J = 6.8 Hz, 3H), 1.07 – 0.92 (m, 3H)。4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(3-amino-2 was purified by Chiral-Prep-HPLC using the following conditions -Chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridino[2,3-d]pyrimidine-2 Atropisomeric mixture of (1H)-ketone (180 mg): Column: CHIRALPAK IF, 2cm × 25cm, 5um; Mobile phase: (Hex:DCM(v/v =3:1)):IPA(v/ v =90:10); detection wavelength: UV 220nm, 81 mg (45%) of 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl) -7-(3-Amino-2-chloro-4,5,6-trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridine [2,3-d]pyrimidin-2(1H)-one (first eluting isomer, compound 7-1) as an off-white solid. LCMS: m/z =644 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.47 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.31 – 7.20 (m, 1H), 6.86 – 6.77 (m , 1H), 6.34 – 6.25 (m, 1H), 5.87 – 5.76 (m, 1H), 4.81 – 4.62 (m, 2H), 4.62 – 4.39 (m, 2H), 3.92 – 3.75 (m, 2H), 2.85 – 2.72 (m, 1H), 2.03 (d, J = 10.7 Hz, 3H), 1.56 – 1.39 (m, 6H), 1.18 (d, J = 6.8 Hz, 3H), 1.04 – 0.95 (m, 3H). and 79 mg (43.89%) of 4-((3S,5R)-4-propenyl-3,5-dimethylpiperazin-1-yl)-7-(3-amino-2-chloro- 4,5,6-Trifluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyridinepara[2,3-d]pyrimidine-2(1H) - Ketone (second eluting isomer, compound 7-2) as an off-white solid. LCMS: m/z =644 [M+1] + . 1 H NMR (400 MHz, CD 3 OD) δ 8.47 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 7.32 – 7.18 (m, 1H), 6.86 – 6.75 (m , 1H), 6.32 – 6.22 (m, 1H), 5.86 – 5.75 (m, 1H), 4.70 (s, 2H), 4.60 – 4.38 (m, 2H), 3.87 – 3.76 (m, 2H), 2.86 – 2.71 (m, 1H), 2.03 (d, J = 10.7 Hz, 3H), 1.60 – 1.42 (m, 6H), 1.18 (d, J = 6.8 Hz, 3H), 1.07 – 0.92 (m, 3H).
如下的化合物可被合成：
藥理實驗Pharmacological experiments
1. SOS1催化的核苷酸交換測試1. SOS1-catalyzed nucleotide exchange assay
將HIS-KRAS (G12C, aa 2-185, Sino biological)用EDTA 緩衝液(20 mM HEPES, pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.01%(v/v) Tween-20)稀釋至5 μM，25 °C條件下孵育30分鐘。用含有120 nM GDP和MAb anti 6HIS-Tb cryptate Gold(Cisbio)的測定緩衝液（25 mM HEPES, pH 7.4, 120 mM NaCl, 5 mM MgCl2
, 1 mM DTT, 0.01% (v/v) Tween 20, 0.1% (w/v) BSA)將EDTA預處理過的HIS-KRAS（G12C）稀釋至12 nM並在25 °C條件下孵育1小時以製備GDP加載的HIS-KRAS（G12C）。將加載的GDP的HIS-KRAS（G12C）與稀釋好的化合物在384孔板（Greiner）中預孵育1小時後，然後將純化的SOS1 ExD(Flag tag, aa 564-1049)和BODIPY™ FL GTP (Invitrogen)加入測試孔（最終濃度：3 nM HIS-KRAS(G12C), 2 μM SOS1 ExD, 80 nM BODIPY™ FL GTP, 21 ng/mL MAb Anti 6HIS-Tb cryptate Gold），然後於25°C孵育4小時。然後使用Tecan Spark多模式酶標儀測定TR-FRET訊號，檢測參數：F486: 激發波長 340 nm，吸收波長 486 nm，延遲時間 100 μs，整合時間 200 μs；F515: 激發波長340 nm，吸收波長 515 nm，延遲時間100 μs，整合時間 200 μs。計算每個孔的TR-FRET比值，TR-FRET比值=（F515訊號/F486訊號）*10000。使用4參數對數模型（4-parameter logistic model）分析數據以計算IC50
值。SOS1催化核苷酸交換測試結果如下表2：
表2
從表2可以看出，本發明代表性化合物在SOS1催化的核苷酸交換實驗下有好的抑制活性，尤其是化合物2-1。化合物2-1的抑制活性可達到對照化合物Amg-510的約2倍。 It can be seen from Table 2 that the representative compounds of the present invention have good inhibitory activity under the SOS1-catalyzed nucleotide exchange assay, especially compound 2-1. The inhibitory activity of compound 2-1 was about 2 times that of the control compound Amg-510.
2. 磷酸化-ERK1/2（THR202/TYR204）HTRF測試2. Phospho-ERK1/2 (THR202/TYR204) HTRF assay
將表達KRAS G12C突變蛋白的NCI-H358細胞在含10%牛胎兒血清（Gibco）的RPMI1640培養基（Gibco）中培養。將培養基中的NCI-H358細胞以40,000個細胞/孔的濃度接種至96孔細胞培養板內，並放置在細胞培養箱內（37℃，5% CO2
）孵育過夜。第二天，將每孔中的培養液移除，並加入以測試培養液（RPMI 1640, 0.1% FBS）稀釋的化合物。在細胞培養箱內（37℃，5% CO2
）孵育2小時後，將96孔板內的培養液移除，然後每孔加入50 μL的1× 細胞裂解液 (Cisbio)，將培養板在25℃震盪孵育45min。將10 μL 細胞裂解液轉移至384孔板 (Greiner)，該384孔板包含2.5 μL/孔 HTRF® 預先混合的抗體 (Cisbio 64AERPEH)，在25℃孵育4小時，然後在Tecan Spark多模式酶標儀中讀取HTRF訊號。使用4參數對數模型（4-parameter logistic model）分析數據以計算IC50
值。磷酸化-ERK1/2（THR202/TYR204）HTRF測試結果如下表3:
表3
從表3可以看出本發明代表性化合物在磷酸化-ERK1/2（THR202/TYR204）HTRF測試下具有好的抑制活性，尤其是化合物2-1。化合物2-1的抑制活性可達到對照化合物Amg-510的約2倍。It can be seen from Table 3 that the representative compounds of the present invention have good inhibitory activity under the phosphorylated-ERK1/2 (THR202/TYR204) HTRF test, especially compound 2-1. The inhibitory activity of compound 2-1 was about 2 times that of the control compound Amg-510.
3. 小鼠藥代動力學研究3. Mouse Pharmacokinetic Study
本研究的目的在於評價按照單劑量給藥的化合物在雌性Balb/c(♀)小鼠體內的藥代動力學性質。小鼠給藥前一天禁食過夜，自由飲水。對於每個化合物需要6只小鼠，並將這6只小鼠分成兩組（n=3/組），A組和B組。A組小鼠給予單次3mg/kg劑量的化合物（iv）。B組小鼠給予單次10mg/kg劑量的化合物（po）。A組小鼠於給藥前及給藥後0.083、0.25、0.5、1、2、4、6、8、24h時間點採集血液樣品。B組小鼠於給藥前及給藥後0.25、0.5、1、2、3、4、6、8、24h時間點採集血液樣品。離心前將血液樣品置於冰上，離心後獲得血漿樣品，在分析前，將該血漿樣品在-80℃儲藏，採用LC-MS/MS方法測定血漿樣品中的化合物的濃度。所得結果如表4所示：
表4
從表4可以看出本發明代表性化合物2和化合物2-1在小鼠模型中具有好的藥代動力學性質，尤其是化合物2-1，相比於Amg-510，其具有高的Cmax 和AUC0-24h ，這使得化合物2-1更適合作為臨床上的口服治療活性成分用於治療KRAS G12C突變的癌症。It can be seen from Table 4 that the representative compound 2 and compound 2-1 of the present invention have good pharmacokinetic properties in the mouse model, especially compound 2-1, which has high C compared to Amg-510 max and AUC 0-24h , which make compound 2-1 more suitable as a clinical oral therapeutic active ingredient for the treatment of KRAS G12C mutated cancers.
4. 犬藥代動力學研究4. Canine Pharmacokinetic Study
本研究的目的在於評價按照單劑量給藥的化合物在比格犬體內的藥代動力學性質。犬給藥前一天禁食過夜，自由飲水。對於每個化合物需要4只比格犬，且將該4只比格犬分成兩組，A組和B組（每組中1只雄性（♂）和1只雌性（♀））。A組比格犬給予單次1mg/kg劑量的化合物（iv）。B組比格犬給予單次3mg/kg劑量的化合物（po）。A組比格犬於給藥前及給藥後0.083、0.25、0.5、1、2、4、6、8和24h時間點採集血液樣品。B組比格犬於給藥前及給藥後0.25、0.5、1、2、4、6、8和24h時間點採集血液樣品。離心前將血液樣品置於冰上，離心後獲得血漿樣品，在分析前，將該血漿樣品在-80℃儲藏，採用LC-MS/MS方法測定血漿樣品中的化合物的濃度。所得結果如表5所示：
表5
從表5可以看出相比於Amg-510，化合物2-1在比格犬模型中具有優良的藥代動力學性質。化合物2-1的Cmax 和AUC0-24h 均顯著高於Amg-510，例如，在雄性比格犬中，化合物2-1的AUC0-24h 可達到對照化合物Amg-510的3倍以上，且在雌性比格犬中，化合物2-1的AUC0-24h 可達到對照化合物Amg-510的12倍以上，這使得化合物2-1更適合作為臨床上的口服治療活性成分用於治療KRAS G12C突變的癌症。It can be seen from Table 5 that compound 2-1 has excellent pharmacokinetic properties in the beagle dog model compared to Amg-510. The Cmax and AUC 0-24h of compound 2-1 were significantly higher than those of Amg-510. For example, in male beagle dogs, the AUC 0-24h of compound 2-1 could reach more than 3 times that of the control compound Amg-510, And in female beagle dogs, the AUC 0-24h of compound 2-1 can reach more than 12 times that of the control compound Amg-510, which makes compound 2-1 more suitable as a clinical oral therapeutic active ingredient for the treatment of KRAS G12C Mutated cancer.
5. 食蟹猴藥代動力學研究5. Pharmacokinetic study in cynomolgus monkeys
本研究的目的在於評價按照單劑量給藥的化合物在食蟹猴體內的藥代動力學性質。猴子給藥前一天禁食過夜，自由飲水。對於每個化合物需要4只猴，且將該4只猴分成兩組，A組和B組（每組中1只雄性（♂）和1只雌性（♀））。A組猴給予單次1 mg/kg劑量的化合物（iv）。B組猴給予單次3 mg/kg劑量的化合物（po）。A組猴於給藥前及給藥後0.083、0.25、0.5、1、2、4、6、8和24h時間點採集血液樣品。B組猴於給藥前及給藥後0.25、0.5、1、2、4、6、8和24h時間點採集血液樣品。離心前將血液樣品置於冰上，離心後獲得血漿樣品，在分析前，將該血漿樣品在-80℃儲藏，採用LC-MS/MS方法測定血漿樣品中的化合物的濃度。所得結果如表6所示：
表6
從表6可以看出相比於Amg-510，化合物2-1在猴模型中具有優良的藥代動力學性質。化合物2-1的Cmax 和AUC0-24h 均顯著高於Amg-510，例如，在雄性食蟹猴中，化合物2-1的AUC0-24h 可達到對照化合物Amg-510的15倍以上，且在雌性食蟹猴中，化合物2-1的AUC可達到對照化合物Amg-510的10倍以上，這使得化合物2-1更適合作為臨床上的口服治療活性成分用於治療KRAS G12C突變的癌症。It can be seen from Table 6 that compound 2-1 has superior pharmacokinetic properties in monkey model compared to Amg-510. The Cmax and AUC 0-24h of compound 2-1 were significantly higher than those of Amg-510. For example, in male cynomolgus monkeys, the AUC 0-24h of compound 2-1 could reach more than 15 times that of the control compound Amg-510, And in female cynomolgus monkeys, the AUC of compound 2-1 can reach more than 10 times that of the control compound Amg-510, which makes compound 2-1 more suitable as a clinical oral therapeutic active ingredient for the treatment of KRAS G12C mutant cancers. .
6. NCI-H1373（KRASG12C ）異種移植模型中的藥效6. Efficacy in NCI-H1373 (KRAS G12C ) Xenograft Model
將5*106
個NCI-H1373（KRASG12C
）細胞與PBS及基質膠（Corning）的混合物（PBS/基質膠=1:1（v/v））皮下接種於雌性BALB/c裸小鼠（6-8周）的右側背部。每天監測小鼠且當腫瘤可見時開始用卡尺測量。腫瘤體積通過測量兩個垂直方向上的直徑使用公式V=(L*W2
)/2計算，其中，L和W分別指腫瘤的長徑和短徑。待平均瘤體積生長至150-200 mm3
時，將小鼠隨機分組(n=6/組)並給藥治療。治療期間(~3周)每週測量兩次腫瘤體積和小鼠體重。腫瘤抑制率通過：TGI% = (1-(Vt - Vt0
) / (Vc - Vc0
)) * 100%計算，其中Vc和Vt分別為實驗結束時對照組和給藥組的平均瘤體積，Vc0
和Vt0
分別為開始給藥時對照組和給藥組的平均瘤體積。結果如下表7和圖1：
表7
從表7和圖1中可以看出化合物2-1在體內具有良好的藥效，且可觀測到腫瘤的消退。 It can be seen from Table 7 and Figure 1 that Compound 2-1 has good efficacy in vivo, and tumor regression can be observed.
7. SW837（KRASG12C ）異種移植模型中安全性探索實驗7. SW837 (KRAS G12C ) Xenograft Model Safety Discovery Experiment
將1*107 個SW837（KRASG12C ）細胞與PBS及基質膠（Corning）的混合物（PBS/基質膠=1:1（v/v））皮下接種於雌性NOD SCID小鼠（6-8周）的右側背部。每天監測小鼠且當腫瘤可見時開始用卡尺測量。腫瘤體積通過測量兩個垂直方向上的直徑使用公式為V=(L*W2 )/2計算，其中，L和W分別指腫瘤的長徑和短徑。在小鼠分組以研究藥效之後，剩餘的小鼠(n=8)被用於探索安全性。小鼠用400mg/kg化合物2-1 (po, QD)治療22天，且在治療期間每週測量兩次小鼠體重。小鼠體重隨細胞植入後的天數的變化顯示在圖2中。從圖2中可以看出化合物2-1有良好的安全性。A mixture of 1*10 7 SW837 (KRAS G12C ) cells with PBS and Matrigel (Corning) (PBS/Matrigel = 1:1 (v/v)) was subcutaneously inoculated into female NOD SCID mice (6-8 weeks). ) on the right back. Mice were monitored daily and calipers started when tumors were visible. Tumor volume was calculated by measuring the diameters in two perpendicular directions using the formula V=(L*W 2 )/2, where L and W refer to the long and short diameters of the tumor, respectively. After mice were grouped to study drug efficacy, the remaining mice (n=8) were used to explore safety. Mice were treated with 400 mg/kg Compound 2-1 (po, QD) for 22 days, and mouse body weights were measured twice weekly during the treatment period. Changes in mouse body weight as a function of days after cell implantation are shown in FIG. 2 . It can be seen from Figure 2 that compound 2-1 has good safety.
應當理解，如果本發明引用了任何現有技術出版物，則應理解為：這樣的引用並不意味著承認該出版物是任何國家本領域公知常識的一部分。儘管為了清楚理解起見，本發明通過示例的方式對上述發明進行了詳細描述，但是對於本領域技術人員顯而易見的某些較小的改變和修改。因此，說明書和實施例不應解釋為限制本發明的範圍。It should be understood that if the present disclosure references any prior art publication, it should be understood that such citation does not imply an admission that such publication is part of the common general knowledge in the field in any country. Although the foregoing invention has been described in detail by way of example for the sake of clarity of understanding, certain minor changes and modifications will be apparent to those skilled in the art. Therefore, the specification and examples should not be construed to limit the scope of the present invention.
無none
圖1為在NCI-H1373（KRASG12C ）異種移植模型中，化合物2-1或溶劑給藥後，腫瘤體積隨接種後的天數的變化圖。 圖2為在SW837（KRASG12C ）異種移植模型中，化合物2-1或溶劑給藥後，體重隨接種後的天數的變化圖。Figure 1 is a graph showing the change in tumor volume with days after inoculation after compound 2-1 or vehicle administration in the NCI-H1373 (KRAS G12C ) xenograft model. Figure 2 is a graph showing the change in body weight with the days after inoculation after compound 2-1 or vehicle administration in the SW837 (KRAS G12C ) xenograft model.
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| MX2022002465A (en) | 2019-08-29 | 2022-05-19 | Mirati Therapeutics Inc | Kras g12d inhibitors. |
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